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Sample records for regulated sphingolipid synthesis

  1. Developmentally Regulated Sphingolipid Synthesis in African Trypanosomes

    PubMed Central

    Sutterwala, Shaheen S.; Hsu, Fong Fu; Sevova, Elitza S.; Schwartz, Kevin J.; Zhang, Kai; Key, Phillip; Turk, John; Beverley, Stephen M.; Bangs, James D.

    2008-01-01

    Sphingolipids are essential components of eukaryotic membranes, and many unicellular eukaryotes, including kinetoplastid protozoa, are thought to synthesize exclusively inositol phosphorylceramide (IPC). Here we characterize sphingolipids from Trypanosoma brucei, and a trypanosome sphingolipid synthase gene family (TbSLS1-4) that is orthologous to Leishmania IPC synthase. Procyclic trypanosomes contain IPC, but also sphingomyelin, while surprisingly bloodstream stage parasites contain sphingomyelin and ethanolamine phosphorylceramide (EPC), but no detectable IPC. In vivo fluorescent ceramide labeling confirmed stage specific biosynthesis of both sphingomyelin and IPC. Expression of TbSLS4 in Leishmania resulted in production of sphingomyelin and EPC suggesting that the TbSLS gene family has bi-functional synthase activity. RNAi silencing of TbSLS1-4 in bloodstream trypanosomes led to rapid growth arrest and eventual cell death. Ceramide levels were increased >3-fold by silencing suggesting a toxic downstream effect mediated by this potent intracellular messenger. Topology predictions support a revised six transmembrane domain model for the kinetoplastid sphingolipid synthases consistent with the proposed mammalian SM synthase structure. This work reveals novel diversity and regulation in sphingolipid metabolism in this important group of human parasites. PMID:18699867

  2. Autophagy regulates sphingolipid levels in the liver.

    PubMed

    Alexaki, Aikaterini; Gupta, Sita D; Majumder, Saurav; Kono, Mari; Tuymetova, Galina; Harmon, Jeffrey M; Dunn, Teresa M; Proia, Richard L

    2014-12-01

    Sphingolipid levels are tightly regulated to maintain cellular homeostasis. During pathologic conditions such as in aging, inflammation, and metabolic and neurodegenerative diseases, levels of some sphingolipids, including the bioactive metabolite ceramide, are elevated. Sphingolipid metabolism has been linked to autophagy, a critical catabolic process in both normal cell function and disease; however, the in vivo relevance of the interaction is not well-understood. Here, we show that blocking autophagy in the liver by deletion of the Atg7 gene, which is essential for autophagosome formation, causes an increase in sphingolipid metabolites including ceramide. We also show that overexpression of serine palmitoyltransferase to elevate de novo sphingolipid biosynthesis induces autophagy in the liver. The results reveal autophagy as a process that limits excessive ceramide levels and that is induced by excessive elevation of de novo sphingolipid synthesis in the liver. Dysfunctional autophagy may be an underlying mechanism causing elevations in ceramide that may contribute to pathogenesis in diseases.

  3. Sphingolipid metabolism regulates development and lifespan in Caenorhabditis elegans

    PubMed Central

    Cutler, Roy G.; Thompson, Kenneth W.; Camandola, Simonetta; Mack, Kendra T.; Mattson, Mark P.

    2015-01-01

    Sphingolipids are a highly conserved lipid component of cell membranes involved in the formation of lipid raft domains that house many of the receptors and cell-to-cell signaling factors involved in regulating cell division, maturation, and terminal differentiation. By measuring and manipulating sphingolipid metabolism using pharmacological and genetic tools in Caenorhabditis elegans, we provide evidence that the synthesis and remodeling of specific ceramides (e.g., dC18:1–C24:1), gangliosides (e.g., GM1–C24:1), and sphingomyelins (e.g., dC18:1–C18:1) influence development rate and lifespan. We found that the levels of fatty acid chain desaturation and elongation in many sphingolipid species increased during development and aging, with no such changes in developmentally-arrested dauer larvae or normal adults after food withdrawal (an anti-aging intervention). Pharmacological inhibitors and small interfering RNAs directed against serine palmitoyl transferase and glucosylceramide synthase acted to slow development rate, extend the reproductive period, and increase lifespan. In contrast, worms fed an egg yolk diet rich in sphingolipids exhibited accelerated development and reduced lifespan. Our findings demonstrate that sphingolipid accumulation and remodeling are critical events that determine development rate and lifespan in the nematode model, with both development rate and aging being accelerated by the synthesis of sphingomyelin, and its metabolism to ceramides and gangliosides. PMID:25437839

  4. Sphingolipids are required for mammalian epidermal barrier function. Inhibition of sphingolipid synthesis delays barrier recovery after acute perturbation.

    PubMed Central

    Holleran, W M; Man, M Q; Gao, W N; Menon, G K; Elias, P M; Feingold, K R

    1991-01-01

    Stratum corneum lipids comprise an approximately equimolar mixture of sphingolipids, cholesterol, and free fatty acids, arranged as intercellular membrane bilayers that are presumed to mediate the epidermal permeability barrier. Prior studies have shown that alterations in epidermal barrier function lead to a rapid increase in cholesterol and fatty acid synthesis which parallels the early stages of the repair process. Despite an abundance of indirect evidence for their role in the barrier, the importance of sphingolipids has yet to be demonstrated directly. Whereas sphingolipid synthesis also increases during barrier repair, this response is delayed in comparison to cholesterol and fatty acid synthesis (Holleran, W.M., et al. 1991. J. Lipid Res. 32:1151-1158). To further delineate the role of sphingolipids in barrier homeostasis, we assessed the impact of inhibition of sphingolipid synthesis on epidermal barrier recovery. A single topical application of beta-chloro-L-alanine (beta-CA), an irreversible inhibitor of serine-palmitoyl transferase (SPT), applied to acetone-treated skin of hairless mice resulted in: (a) greater than 75% inhibition of SPT activity at 30 min (P less than 0.001); (b) a global decrease in sphingolipid synthesis between 1 and 3 h (P less than 0.02); (c) reduction of epidermal sphingolipid content at 18 h (P less than 0.01); (d) delayed reaccumulation of histochemical staining for sphingolipids in the stratum corneum; and (e) reduced numbers and contents of lamellar bodies in the stratum granulosum. Finally, despite its immediate, marked diminution of sphingolipid synthesis, beta-CA slowed barrier recovery only at late time points (greater than 6 h) after acetone treatment. This inhibition was overridden by coapplications of ceramides (the distal SPT product), indicating that the delay in repair was not due to non-specific toxicity. These studies demonstrate a distinctive role for epidermal sphingolipids in permeability barrier homeostasis

  5. Regulation of PP2A by Sphingolipid Metabolism and Signaling

    PubMed Central

    Oaks, Joshua; Ogretmen, Besim

    2014-01-01

    Protein phosphatase 2A (PP2A) is a serine/threonine phosphatase that is a primary regulator of cellular proliferation through targeting of proliferative kinases, cell cycle regulators, and apoptosis inhibitors. It is through the regulation of these regulatory elements that gives PP2A tumor suppressor functions. In addition to mutations on the regulatory subunits, the phosphatase/tumor suppressing activity of PP2A is also inhibited in several cancer types due to overexpression or modification of the endogenous PP2A inhibitors such as SET/I2PP2A. This review focuses on the current literature regarding the interactions between the lipid signaling molecules, selectively sphingolipids, and the PP2A inhibitor SET for the regulation of PP2A, and the therapeutic potential of sphingolipids as PP2A activators for tumor suppression via targeting SET oncoprotein. PMID:25642418

  6. Nogo-B regulates endothelial sphingolipid homeostasis to control vascular function and blood pressure

    PubMed Central

    Kothiya, Milankumar; Galvani, Sylvain; Obinata, Hideru; Bucci, Mariarosaria; Giordano, Frank J; Jiang, Xian-Cheng; Hla, Timothy; Di Lorenzo, Annarita

    2015-01-01

    Endothelial dysfunction is a critical factor in many cardiovascular diseases, including hypertension. Although lipid signaling has been implicated in endothelial dysfunction and cardiovascular disease, specific molecular mechanisms are poorly understood. Here we report that Nogo-B, a membrane protein of the endoplasmic reticulum, regulates endothelial sphingolipid biosynthesis with direct effects on vascular function and blood pressure. Nogo-B inhibits serine palmitoyltransferase, the rate-limiting enzyme of the de novo sphingolipid biosynthetic pathway, thereby controlling production of endothelial sphingosine 1-phosphate and autocrine, G protein–coupled receptor–dependent signaling by this metabolite. Mice lacking Nogo-B either systemically or specifically in endothelial cells are hypotensive, resistant to angiotensin II–induced hypertension and have preserved endothelial function and nitric oxide release. In mice that lack Nogo-B, pharmacological inhibition of serine palmitoyltransferase with myriocin reinstates endothelial dysfunction and angiotensin II–induced hypertension. Our study identifies Nogo-B as a key inhibitor of local sphingolipid synthesis and shows that autocrine sphingolipid signaling within the endothelium is critical for vascular function and blood pressure homeostasis. PMID:26301690

  7. Sphingolipid regulation of ezrin, radixin, and moesin proteins family: implications for cell dynamics.

    PubMed

    Adada, Mohamad; Canals, Daniel; Hannun, Yusuf A; Obeid, Lina M

    2014-05-01

    A key but poorly studied domain of sphingolipid functions encompasses endocytosis, exocytosis, cellular trafficking, and cell movement. Recently, the ezrin, radixin and moesin (ERM) family of proteins emerged as novel potent targets regulated by sphingolipids. ERMs are structural proteins linking the actin cytoskeleton to the plasma membrane, also forming a scaffold for signaling pathways that are used for cell proliferation, migration and invasion, and cell division. Opposing functions of the bioactive sphingolipid ceramide and sphingosine-1-phosphate (S1P), contribute to ERM regulation. S1P robustly activates whereas ceramide potently deactivates ERM via phosphorylation/dephosphorylation, respectively. This recent dimension of cytoskeletal regulation by sphingolipids opens up new avenues to target cell dynamics, and provides further understanding of some of the unexplained biological effects mediated by sphingolipids. In addition, these studies are providing novel inroads into defining basic mechanisms of regulation and action of bioactive sphingolipids. This review describes the current understanding of sphingolipid regulation of the cytoskeleton, it also describes the biologies in which ERM proteins have been involved, and finally how these two large fields have started to converge. This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology.

  8. Sphingolipids: Key Regulators of Apoptosis and Pivotal Players in Cancer Drug Resistance

    PubMed Central

    Giussani, Paola; Tringali, Cristina; Riboni, Laura; Viani, Paola; Venerando, Bruno

    2014-01-01

    Drug resistance elicited by cancer cells still constitutes a huge problem that frequently impairs the efficacy of both conventional and novel molecular therapies. Chemotherapy usually acts to induce apoptosis in cancer cells; therefore, the investigation of apoptosis control and of the mechanisms used by cancer cells to evade apoptosis could be translated in an improvement of therapies. Among many tools acquired by cancer cells to this end, the de-regulated synthesis and metabolism of sphingolipids have been well documented. Sphingolipids are known to play many structural and signalling roles in cells, as they are involved in the control of growth, survival, adhesion, and motility. In particular, in order to increase survival, cancer cells: (a) counteract the accumulation of ceramide that is endowed with pro-apoptotic potential and is induced by many drugs; (b) increase the synthesis of sphingosine-1-phosphate and glucosylceramide that are pro-survivals signals; (c) modify the synthesis and the metabolism of complex glycosphingolipids, particularly increasing the levels of modified species of gangliosides such as 9-O acetylated GD3 (αNeu5Ac(2-8)αNeu5Ac(2-3)βGal(1-4)βGlc(1-1)Cer) or N-glycolyl GM3 (αNeu5Ac (2-3)βGal(1-4)βGlc(1-1)Cer) and de-N-acetyl GM3 (NeuNH(2)βGal(1-4)βGlc(1-1)Cer) endowed with anti-apoptotic roles and of globoside Gb3 related to a higher expression of the multidrug resistance gene MDR1. In light of this evidence, the employment of chemical or genetic approaches specifically targeting sphingolipid dysregulations appears a promising tool for the improvement of current chemotherapy efficacy. PMID:24625663

  9. Sphingolipids regulate telomere clustering by affecting the transcription of genes involved in telomere homeostasis.

    PubMed

    Ikeda, Atsuko; Muneoka, Tetsuya; Murakami, Suguru; Hirota, Ayaka; Yabuki, Yukari; Karashima, Takefumi; Nakazono, Kota; Tsuruno, Masahiro; Pichler, Harald; Shirahige, Katsuhiko; Kodama, Yukiko; Shimamoto, Toshi; Mizuta, Keiko; Funato, Kouichi

    2015-07-15

    In eukaryotic organisms, including mammals, nematodes and yeasts, the ends of chromosomes, telomeres are clustered at the nuclear periphery. Telomere clustering is assumed to be functionally important because proper organization of chromosomes is necessary for proper genome function and stability. However, the mechanisms and physiological roles of telomere clustering remain poorly understood. In this study, we demonstrate a role for sphingolipids in telomere clustering in the budding yeast Saccharomyces cerevisiae. Because abnormal sphingolipid metabolism causes downregulation of expression levels of genes involved in telomere organization, sphingolipids appear to control telomere clustering at the transcriptional level. In addition, the data presented here provide evidence that telomere clustering is required to protect chromosome ends from DNA-damage checkpoint signaling. As sphingolipids are found in all eukaryotes, we speculate that sphingolipid-based regulation of telomere clustering and the protective role of telomere clusters in maintaining genome stability might be conserved in eukaryotes.

  10. Tamoxifen regulation of sphingolipid metabolism--Therapeutic implications.

    PubMed

    Morad, Samy A F; Cabot, Myles C

    2015-09-01

    Tamoxifen, a triphenylethylene antiestrogen and one of the first-line endocrine therapies used to treat estrogen receptor-positive breast cancer, has a number of interesting, off-target effects, and among these is the inhibition of sphingolipid metabolism. More specifically, tamoxifen inhibits ceramide glycosylation, and enzymatic step that can adventitiously support the influential tumor-suppressor properties of ceramide, the aliphatic backbone of sphingolipids. Additionally, tamoxifen and metabolites N-desmethyltamoxifen and 4-hydroxytamoxifen, have been shown to inhibit ceramide hydrolysis by the enzyme acid ceramidase. This particular intervention slows ceramide destruction and thereby depresses formation of sphingosine 1-phosphate, a mitogenic sphingolipid with cancer growth-promoting properties. As ceramide-centric therapies are becoming appealing clinical interventions in the treatment of cancer, agents like tamoxifen that can retard the generation of mitogenic sphingolipids and buffer ceramide clearance via inhibition of glycosylation, take on new importance. In this review, we present an abridged, lay introduction to sphingolipid metabolism, briefly chronicle tamoxifen's history in the clinic, examine studies that demonstrate the impact of triphenylethylenes on sphingolipid metabolism in cancer cells, and canvass works relevant to the use of tamoxifen as adjuvant to drive ceramide-centric therapies in cancer treatment. The objective is to inform the readership of what could be a novel, off-label indication of tamoxifen and structurally-related triphenylethylenes, an indication divorced from estrogen receptor status and one with application in drug resistance.

  11. Tamoxifen regulation of sphingolipid metabolism—therapeutic implications

    PubMed Central

    Morad, Samy A F; Cabot, Myles C

    2015-01-01

    Tamoxifen, a triphenylethylene antiestrogen and one of the first-line endocrine therapies used to treat estrogen receptor-positive breast cancer, has a number of interesting, off-target effects, and among these is the inhibition of sphingolipid metabolism. More specifically, tamoxifen inhibits ceramide glycosylation, and enzymatic step that can adventitiously support the influential tumor-suppressor properties of ceramide, the aliphatic backbone of sphingolipids. Additionally, tamoxifen and metabolites N-desmethyltamoxifen and 4-hydroxytamoxifen, have been shown to inhibit ceramide hydrolysis by the enzyme acid ceramidase. This particular intervention slows ceramide destruction and thereby depresses formation of sphingosine 1-phosphate, a mitogenic sphingolipid with cancer growth-promoting properties. As ceramide-centric therapies are becoming appealing clinical interventions in the treatment of cancer, agents like tamoxifen that can retard the generation of mitogenic sphingolipids and buffer ceramide clearance via inhibition of glycosylation, take on new importance. In this review, we present an abridged, lay introduction to sphingolipid metabolism, briefly chronicle tamoxifen’s history in the clinic, examine studies that demonstrate the impact of triphenylethylenes on sphingolipid metabolism in cancer cells, and canvass works relevant to the use of tamoxifen as adjuvant to drive ceramide-centric therapies in cancer treatment. The objective is to inform the readership of what could be a novel, off-label indication of tamoxifen and structurally-related triphenylethylenes, an indication divorced from estrogen receptor status and one with application in drug resistance. PMID:25964209

  12. Sphingolipids as Regulators of the Phagocytic Response to Fungal Infections

    PubMed Central

    Bryan, Arielle M.; Del Poeta, Maurizio; Luberto, Chiara

    2015-01-01

    Fungal infections pose a significant risk for the increasing population of individuals who are immunocompromised. Phagocytes play an important role in immune defense against fungal pathogens, but the interactions between host and fungi are still not well understood. Sphingolipids have been shown to play an important role in many cell functions, including the function of phagocytes. In this review, we discuss major findings that relate to the importance of sphingolipids in macrophage and neutrophil function and the role of macrophages and neutrophils in the most common types of fungal infections, as well as studies that have linked these three concepts to show the importance of sphingolipid signaling in immune response to fungal infections. PMID:26688618

  13. Synthesis of Sphingolipids Impacts Survival of Porphyromonas gingivalis and the Presentation of Surface Polysaccharides

    PubMed Central

    Moye, Zachary D.; Valiuskyte, Kornelija; Dewhirst, Floyd E.; Nichols, Frank C.; Davey, Mary E.

    2016-01-01

    Bacteria alter the biophysical properties of their membrane lipids in response to environmental cues, such as shifts in pH or temperature. In essence, lipid composition determines membrane structure, which in turn influences many basic functions, such as transport, secretion, and signaling. Like other members of the phylum Bacteroidetes, the oral anaerobe Porphyromonas gingivalis possesses the ability to synthesize a variety of novel membrane lipids, including species of dihydroceramides that are distinct, yet similar in structure to sphingolipids produced by the human host. The role of dihydroceramides in the physiology and pathogenic potential of the human microbiota is only beginning to be explored; yet there is increasing data indicating that these lipids play a role in human diseases, such as periodontitis and multiple sclerosis. Here, we report on the identification of a gene (PG1780) in the chromosome of P. gingivalis strain W83 encoding a putative serine palmitoyltransferase, the enzyme that catalyzes the first step in sphingolipid biosynthesis. While we were able to detect dihydroceramides in whole lipid extracts of P. gingivalis cells as well as crude preparations of outer membrane vesicles, sphingolipids were absent in the PG1780 mutant strain. Moreover, we show that the synthesis of sphingolipids plays an essential role in the long-term survival of the organism as well as its resistance to oxidative stress. Further, a PG1780 mutant displayed much lower activity of cell-associated arginine and lysine gingipains, yet slightly higher activity in the corresponding culture supernates, which we hypothesize is due to altered membrane properties and anchoring of these proteases to the cell surface. In addition, we determined that sphingolipid production is critical to the presentation of surface polysaccharides, with the mutant strain displaying less K-antigen capsule and more anionic polysaccharide (APS). Overall, we have discovered that, in addition to their

  14. Sphingolipids in High Fat Diet and Obesity-Related Diseases

    PubMed Central

    Choi, Songhwa; Snider, Ashley J.

    2015-01-01

    Nutrient oversupply associated with a high fat diet (HFD) significantly alters cellular metabolism, and specifically including sphingolipid metabolism. Sphingolipids are emerging as bioactive lipids that play key roles in regulating functions, in addition to their traditional roles as membrane structure. HFD enhances de novo sphingolipid synthesis and turnover of sphingolipids via the salvage pathway, resulting in the generation of ceramide, and more specifically long chain ceramide species. Additionally, HFD elevates sphingomyelin and sphingosine-1 phosphate (S1P) levels in several tissues including liver, skeletal muscle, adipose tissue, and cardiovascular tissues. HFD-stimulated sphingolipid generation contributes to systemic insulin resistance, dysregulated lipid accumulation, and cytokine expression and secretion from skeletal muscle and adipose tissues, exacerbating obesity-related conditions. Furthermore, altered sphingolipid levels, particularly ceramide and sphingomyelin, are involved in obesity-induced endothelial dysfunction and atherosclerosis. In this review, HFD-mediated sphingolipid metabolism and its impact on HFD-induced biology and pathobiology will be discussed. PMID:26648664

  15. Effects of inhibitors of key enzymes of sphingolipid metabolism on insulin-induced glucose uptake and glycogen synthesis in liver cells of old rats.

    PubMed

    Babenko, N A; Kharchenko, V S

    2015-01-01

    Sphingolipids play an important role in the development of insulin resistance. Ceramides are the most potent inhibitors of insulin signal transduction. Ceramides are generated in response to stress stimuli and in old age. In this work, we studied the possible contribution of different pathways of sphingolipid metabolism in age-dependent insulin resistance development in liver cells. Inhibition of key enzymes of sphingolipid synthesis (serine palmitoyl transferase, ceramide synthase) and degradation (neutral and acidic SMases) by means of specific inhibitors (myriocin, fumonisin B1, imipramine, and GW4869) was followed with the reduction of ceramide level and partly improved insulin regulation of glucose metabolism in "old" hepatocytes. Imipramine and GW4869 decreased significantly the acidic and neutral SMase activities, respectively. Treatment of "old" cells with myriocin or fumonisin B1 reduced the elevated in old age ceramide and SM synthesis. Ceramide and SM levels and glucose metabolism regulation by insulin could be improved with concerted action of all tested inhibitors of sphingolipid turnover on hepatocytes. The data demonstrate that not only newly synthesized ceramide and SM but also neutral and acidic SMase-dependent ceramide accumulation plays an important role in development of age-dependent insulin resistance.

  16. Endothelial Nogo-B regulates sphingolipid biosynthesis to promote pathological cardiac hypertrophy during chronic pressure overload

    PubMed Central

    Huang, Yan; Azevedo, Paula S.; Siragusa, Mauro; Bielawski, Jacek; Giordano, Frank J.

    2016-01-01

    We recently discovered that endothelial Nogo-B, a membrane protein of the ER, regulates vascular function by inhibiting the rate-limiting enzyme, serine palmitoyltransferase (SPT), in de novo sphingolipid biosynthesis. Here, we show that endothelium-derived sphingolipids, particularly sphingosine-1-phosphate (S1P), protect the heart from inflammation, fibrosis, and dysfunction following pressure overload and that Nogo-B regulates this paracrine process. SPT activity is upregulated in banded hearts in vivo as well as in TNF-α–activated endothelium in vitro, and loss of Nogo removes the brake on SPT, increasing local S1P production. Hence, mice lacking Nogo-B, systemically or specifically in the endothelium, are resistant to the onset of pathological cardiac hypertrophy. Furthermore, pharmacological inhibition of SPT with myriocin restores permeability, inflammation, and heart dysfunction in Nogo-A/B–deficient mice to WT levels, whereas SEW2871, an S1P1 receptor agonist, prevents myocardial permeability, inflammation, and dysfunction in WT banded mice. Our study identifies a critical role of endothelial sphingolipid biosynthesis and its regulation by Nogo-B in the development of pathological cardiac hypertrophy and proposes a potential therapeutic target for the attenuation or reversal of this clinical condition. PMID:27158676

  17. Hepatic fatty acid uptake is regulated by the sphingolipid acyl chain length

    PubMed Central

    Park, Woo-Jae; Park, Joo-Won; Merrill, Alfred H.; Storch, Judith; Pewzner-Jung, Yael; Futerman, Anthony H.

    2015-01-01

    Ceramide synthase 2 (CerS2) null mice cannot synthesize very-long acyl chain (C22-C24) ceramides resulting in significant alterations in the acyl chain composition of sphingolipids. We now demonstrate that hepatic triacylglycerol (TG) levels are reduced in liver but not in adipose tissue or skeletal muscle in the CerS2 null mouse, both before and after feeding with a high fat diet (HFD), where no weight gain was observed and large hepatic nodules appeared. Uptake of both BODIPY-palmitate and [3H]-palmitate were also abrogated in hepatocytes and liver. The role of a number of key proteins involved in fatty acid uptake was examined, including FATP5, CD36/FAT, FABPpm and cytoplasmic FABP1. Levels of FATP5 and FABP1 were decreased in CerS2 null mouse liver, whereas CD36/FAT levels were significantly elevated and CD36/FAT was also mislocalized upon insulin treatment. Moreover, treatment of hepatocytes with C22-C24-ceramides down-regulated CD36/FAT levels. Infection of CerS2 null mice with recombinant adeno-associated virus (rAAV)-CerS2 restored normal TG levels and corrected the mislocalization of CD36/FAT, but had no effect on the intracellular localization or levels of FATP5 or FABP1. Together, these results demonstrate that hepatic fatty acid uptake via CD36/FAT can be regulated by altering the acyl chain composition of sphingolipids. PMID:25241943

  18. Arabidopsis mutants in sphingolipid synthesis as tools to understand the structure and function of membrane microdomains in plasmodesmata

    PubMed Central

    González-Solís, Ariadna; Cano-Ramírez, Dora L.; Morales-Cedillo, Francisco; Tapia de Aquino, Cinthya; Gavilanes-Ruiz, Marina

    2013-01-01

    Plasmodesmata—intercellular channels that communicate adjacent cells—possess complex membranous structures. Recent evidences indicate that plasmodesmata contain membrane microdomains. In order to understand how these submembrane regions collaborate to plasmodesmata function, it is necessary to characterize their size, composition and dynamics. An approach that can shed light on these microdomain features is based on the use of Arabidopsis mutants in sphingolipid synthesis. Sphingolipids are canonical components of microdomains together with sterols and some glycerolipids. Moreover, sphingolipids are transducers in pathways that display programmed cell death as a defense mechanism against pathogens. The study of Arabidopsis mutants would allow determining which structural features of the sphingolipids are important for the formation and stability of microdomains, and if defense signaling networks using sphingoid bases as second messengers are associated to plasmodesmata operation. Such studies need to be complemented by analysis of the ultrastructure and the use of protein probes for plasmodesmata microdomains and may constitute a very valuable source of information to analyze these membrane structures. PMID:24478783

  19. The ATP-binding cassette transporter-2 (ABCA2) regulates esterification of plasma membrane cholesterol by modulation of sphingolipid metabolism.

    PubMed

    Davis, Warren

    2014-01-01

    The ATP-binding cassette transporters are a large family (~48 genes divided into seven families A-G) of proteins that utilize the energy of ATP-hydrolysis to pump substrates across lipid bilayers against a concentration gradient. The ABC "A" subfamily is comprised of 13 members and transport sterols, phospholipids and bile acids. ABCA2 is the most abundant ABC transporter in human and rodent brain with highest expression in oligodendrocytes, although it is also expressed in neurons. Several groups have studied a possible connection between ABCA2 and Alzheimer's disease as well as early atherosclerosis. ABCA2 expression levels have been associated with changes in cholesterol and sphingolipid metabolism. In this paper, we hypothesized that ABCA2 expression level may regulate esterification of plasma membrane-derived cholesterol by modulation of sphingolipid metabolism. ABCA2 overexpression in N2a neuroblastoma cells was associated with an altered bilayer distribution of the sphingolipid ceramide that inhibited acylCoA:cholesterol acyltransferase (ACAT) activity and cholesterol esterification. In contrast, depletion of endogenous ABCA2 in the rat schwannoma cell line D6P2T increased esterification of plasma membrane cholesterol following treatment with exogenous bacterial sphingomyelinase. These findings suggest that control of ABCA2 expression level may be a key locus of regulation for esterification of plasma membrane-derived cholesterol through modulation of sphingolipid metabolism.

  20. Loss of Frataxin induces iron toxicity, sphingolipid synthesis, and Pdk1/Mef2 activation, leading to neurodegeneration

    PubMed Central

    Chen, Kuchuan; Lin, Guang; Haelterman, Nele A; Ho, Tammy Szu-Yu; Li, Tongchao; Li, Zhihong; Duraine, Lita; Graham, Brett H; Jaiswal, Manish; Yamamoto, Shinya; Rasband, Matthew N; Bellen, Hugo J

    2016-01-01

    Mutations in Frataxin (FXN) cause Friedreich’s ataxia (FRDA), a recessive neurodegenerative disorder. Previous studies have proposed that loss of FXN causes mitochondrial dysfunction, which triggers elevated reactive oxygen species (ROS) and leads to the demise of neurons. Here we describe a ROS independent mechanism that contributes to neurodegeneration in fly FXN mutants. We show that loss of frataxin homolog (fh) in Drosophila leads to iron toxicity, which in turn induces sphingolipid synthesis and ectopically activates 3-phosphoinositide dependent protein kinase-1 (Pdk1) and myocyte enhancer factor-2 (Mef2). Dampening iron toxicity, inhibiting sphingolipid synthesis by Myriocin, or reducing Pdk1 or Mef2 levels, all effectively suppress neurodegeneration in fh mutants. Moreover, increasing dihydrosphingosine activates Mef2 activity through PDK1 in mammalian neuronal cell line suggesting that the mechanisms are evolutionarily conserved. Our results indicate that an iron/sphingolipid/Pdk1/Mef2 pathway may play a role in FRDA. DOI: http://dx.doi.org/10.7554/eLife.16043.001 PMID:27343351

  1. Identification of a New Class of Antifungals Targeting the Synthesis of Fungal Sphingolipids

    PubMed Central

    Mor, Visesato; Rella, Antonella; Farnoud, Amir M.; Singh, Ashutosh; Munshi, Mansa; Bryan, Arielle; Naseem, Shamoon; Konopka, James B.; Ojima, Iwao; Bullesbach, Erika; Ashbaugh, Alan; Linke, Michael J.; Cushion, Melanie; Collins, Margaret; Ananthula, Hari Krishna; Sallans, Larry; Desai, Pankaj B.; Wiederhold, Nathan P.; Fothergill, Annette W.; Kirkpatrick, William R.; Patterson, Thomas; Wong, Lai Hong; Sinha, Sunita; Giaever, Guri; Nislow, Corey; Flaherty, Patrick; Pan, Xuewen; Cesar, Gabriele Vargas; de Melo Tavares, Patricia; Frases, Susana; Miranda, Kildare; Rodrigues, Marcio L.; Luberto, Chiara; Nimrichter, Leonardo

    2015-01-01

    ABSTRACT Recent estimates suggest that >300 million people are afflicted by serious fungal infections worldwide. Current antifungal drugs are static and toxic and/or have a narrow spectrum of activity. Thus, there is an urgent need for the development of new antifungal drugs. The fungal sphingolipid glucosylceramide (GlcCer) is critical in promoting virulence of a variety of human-pathogenic fungi. In this study, we screened a synthetic drug library for compounds that target the synthesis of fungal, but not mammalian, GlcCer and found two compounds [N′-(3-bromo-4-hydroxybenzylidene)-2-methylbenzohydrazide (BHBM) and its derivative, 3-bromo-N′-(3-bromo-4-hydroxybenzylidene) benzohydrazide (D0)] that were highly effective in vitro and in vivo against several pathogenic fungi. BHBM and D0 were well tolerated in animals and are highly synergistic or additive to current antifungals. BHBM and D0 significantly affected fungal cell morphology and resulted in the accumulation of intracellular vesicles. Deep-sequencing analysis of drug-resistant mutants revealed that four protein products, encoded by genes APL5, COS111, MKK1, and STE2, which are involved in vesicular transport and cell cycle progression, are targeted by BHBM. PMID:26106079

  2. Ceramide synthases at the centre of sphingolipid metabolism and biology.

    PubMed

    Mullen, Thomas D; Hannun, Yusuf A; Obeid, Lina M

    2012-02-01

    Sphingolipid metabolism in metazoan cells consists of a complex interconnected web of numerous enzymes, metabolites and modes of regulation. At the centre of sphingolipid metabolism reside CerSs (ceramide synthases), a group of enzymes that catalyse the formation of ceramides from sphingoid base and acyl-CoA substrates. From a metabolic perspective, these enzymes occupy a unique niche in that they simultaneously regulate de novo sphingolipid synthesis and the recycling of free sphingosine produced from the degradation of pre-formed sphingolipids (salvage pathway). Six mammalian CerSs (CerS1-CerS6) have been identified. Unique characteristics have been described for each of these enzymes, but perhaps the most notable is the ability of individual CerS isoforms to produce ceramides with characteristic acyl-chain distributions. Through this control of acyl-chain length and perhaps in a compartment-specific manner, CerSs appear to regulate multiple aspects of sphingolipid-mediated cell and organismal biology. In the present review, we discuss the function of CerSs as critical regulators of sphingolipid metabolism, highlight their unique characteristics and explore the emerging roles of CerSs in regulating programmed cell death, cancer and many other aspects of biology.

  3. Sphingolipids regulate [Mg2+]o uptake and [Mg2+]i content in vascular smooth muscle cells: potential mechanisms and importance to membrane transport of Mg2+.

    PubMed

    Zheng, Tao; Li, Wenyan; Altura, Bella T; Shah, Nilank C; Altura, Burton M

    2011-02-01

    Sphingolipids have a variety of important signaling roles in mammalian cells. We tested the hypothesis that certain sphingolipids and neutral sphingomyelinase (N-SMase) can regulate intracellular free magnesium ions ([Mg2+]i) in vascular smooth muscle (VSM) cells. Herein, we show that several sphingolipids, including C2-ceramide, C8-ceramide, C16-ceramide, and sphingosine, as well as N-SMase, have potent and direct effects on content and mobilization of [Mg2+]i in primary cultured rat aortic smooth muscle cells. All of these sphingolipid molecules increase, rapidly, [Mg2+]i in these vascular cells in a concentration-dependent manner. The increments of [Mg2+]i, induced by these agents, are derived from influx of extracellular Mg2+ and are extracellular Ca2+ concentration-dependent. Phospholipase C and Ca2+/calmodulin/Ca2+-ATPase activity appear to be important in the sphingolipid-induced rises of [Mg2+]i. Activation of certain PKC isozymes may also be required for sphingolipid-induced rises in [Mg2+]i. These novel results suggest that sphingolipids may be homeostatic regulators of extracellular Mg2+ concentration influx (and transport) and [Mg2+]i content in vascular muscle cells.

  4. Amyotrophic lateral sclerosis and denervation alter sphingolipids and up-regulate glucosylceramide synthase

    PubMed Central

    Henriques, Alexandre; Croixmarie, Vincent; Priestman, David A.; Rosenbohm, Angela; Dirrig-Grosch, Sylvie; D'Ambra, Eleonora; Huebecker, Mylene; Hussain, Ghulam; Boursier-Neyret, Claire; Echaniz-Laguna, Andoni; Ludolph, Albert C.; Platt, Frances M.; Walther, Bernard; Spedding, Michael; Loeffler, Jean-Philippe; Gonzalez De Aguilar, Jose-Luis

    2015-01-01

    Amyotrophic lateral sclerosis (ALS) is a fatal adult-onset disease characterized by upper and lower motor neuron degeneration, muscle wasting and paralysis. Growing evidence suggests a link between changes in lipid metabolism and ALS. Here, we used UPLC/TOF-MS to survey the lipidome in SOD1(G86R) mice, a model of ALS. Significant changes in lipid expression were evident in spinal cord and skeletal muscle before overt neuropathology. In silico analysis also revealed appreciable changes in sphingolipids including ceramides and glucosylceramides (GlcCer). HPLC analysis showed increased amounts of GlcCer and downstream glycosphingolipids (GSLs) in SOD1(G86R) muscle compared with wild-type littermates. Glucosylceramide synthase (GCS), the enzyme responsible for GlcCer biosynthesis, was up-regulated in muscle of SOD1(G86R) mice and ALS patients, and in muscle of wild-type mice after surgically induced denervation. Conversely, inhibition of GCS in wild-type mice, following transient peripheral nerve injury, reversed the overexpression of genes in muscle involved in oxidative metabolism and delayed motor recovery. GCS inhibition in SOD1(G86R) mice also affected the expression of metabolic genes and induced a loss of muscle strength and morphological deterioration of the motor endplates. These findings suggest that GSLs may play a critical role in ALS muscle pathology and could lead to the identification of new therapeutic targets. PMID:26483191

  5. Sphingolipids in parasitic protozoa

    PubMed Central

    Zhang, Kai; Bangs, James D.; Beverley, Stephen M.

    2009-01-01

    The surface of most protozoan parasites relies heavily upon lipid-anchored molecules, to form protective barriers and play critical functions required for infectivity. Sphingolipids (SLs) play important roles through their abundance and involvement in membrane microdomain formation, as well as serving as the lipid anchor for many of these molecules, and in some but possibly not all species, as important signaling molecules. Interactions of parasite sphingolipid metabolism with that of the host may potentially contribute to parasite survival and/or host defense. In this chapter we summarize current knowledge of SL structure, synthesis and function in several of the major parasitic protozoan groups. PMID:20919659

  6. Sterols and sphingolipids: Dynamic duo or partners in crime?

    PubMed Central

    Gulati, Sonia; Liu, Ying; Munkacsi, Andrew B.; Wilcox, Lisa; Sturley, Stephen L.

    2010-01-01

    One manner in which eukaryotic cells respond to their environments is by optimizing the composition and proportions of sterols and sphingolipids in membranes. The physical association of the planar ring of sterols with the acyl chains of phospholipids, particularly sphingolipids, produces membrane micro-heterogeneity that is exploited to coordinate several crucial pathways. We hypothesize that these lipid molecules play an integrated role in human disease; when one of the partners is mis-regulated, pathology frequently ensues. Sterols and sphingolipid levels are not coordinated by the action of a single master regulator, however the cross talk between their metabolic pathways is considerable. We describe our perspectives on the key components of synthesis, catabolism and transport of these lipid partners with an emphasis on evolutionarily conserved reactions that produce disease states when defective. PMID:20362613

  7. Molecular facets of sphingolipids: mediators of diseases.

    PubMed

    Ozbayraktar, Fatma Betul Kavun; Ulgen, Kutlu O

    2009-07-01

    Sphingolipids constitute a biologically active lipid class that is significantly important from both structural and regulatory aspects. The manipulation of sphingolipid metabolism is currently being studied as a novel strategy for cancer therapy. The basics of this therapeutic approach lie in the regulation property of sphingolipids on cellular processes, which are important in a cell's fate, such as cell proliferation, apoptosis, cell cycle arrest, senescence, and inflammation. Furthermore, the mutations in the enzymes catalyzing some specific reactions in the sphingolipid metabolism cause mortal lysosomal storage diseases like Fabry, Gaucher, Niemann-Pick, Farber, Krabbe, and Metachromatic Leukodystrophy. Therefore, the alteration of the sphingolipid metabolic pathway determines the choice between life and death. Understanding the sphingolipid metabolism and regulation is significant for the development of new therapeutic approaches for all sphingolipid-related diseases, as well as for cancer. An important feature of the sphingolipid metabolic pathway is the compartmentalization into endoplasmic reticulum, the Golgi apparatus, lysosome and plasma membrane, and this compartmentalization makes the transport of sphingolipids critical for proper functioning. This paper focuses on the structures, metabolic pathways, localization, transport mechanisms, and diseases of sphingolipids in Saccharomyces cerevisiae and humans, and provides the latest comprehensive information on sphingolipid research.

  8. Sphingolipids in spinal cord injury

    PubMed Central

    Jones, Zachary B; Ren, Yi

    2016-01-01

    Spinal cord injury (SCI) is a debilitating condition that affects millions of individuals worldwide. Despite progress over the last few decades, the molecular mechanisms of secondary SCI that continue to occur days and weeks after the original trauma remain poorly understood. As a result, current therapies for SCI are only marginally effective. Sphingolipids, a diverse class of bioactive lipids, have been shown to regulate SCI repair and key secondary injury processes such as apoptosis, ischemia and inflammation. This review will discuss the numerous roles of sphingolipids and highlight the potential of sphingolipid-targeted therapies for SCI. PMID:27570580

  9. Sphingolipids from a symbiotic microbe regulate homeostasis of host intestinal natural killer T cells

    PubMed Central

    An, Dingding; Oh, Sungwhan F.; Olszak, Torsten; Neves, Joana F.; Avci, Fikri; Erturk-Hasdemir, Deniz; Lu, Xi; Zeissig, Sebastian; Blumberg, Richard S.; Kasper, Dennis L.

    2014-01-01

    Summary Co-evolution of beneficial microorganisms with the mammalian intestine fundamentally shapes mammalian physiology. Herein we report that the intestinal microbe Bacteroides fragilis modifies the homeostasis of host invariant natural killer T (iNKT) cells by supplementing the host’s endogenous lipid antigen milieu with unique inhibitory sphingolipids. The process occurs early in life and effectively impedes iNKT cell proliferation during neonatal development. Consequently, total colonic iNKT cell numbers are restricted into adulthood and hosts are protected against experimental iNKT cell–mediated, oxazolone-induced colitis. In studies with neonatal mice lacking access to bacterial sphingolipids, we found that treatment with B. fragilis glycosphingolipids—exemplified by an isolated peak (M.W.=717.6) called GSL-Bf717—reduces colonic iNKT cell numbers and confers protection against oxazolone-induced colitis in adulthood. Our results suggest that the distinctive inhibitory capacity of GSL-Bf717 and similar molecules may prove useful in the treatment of autoimmune and allergic disorders in which iNKT cell activation is destructive. PMID:24439373

  10. The sphingolipid long-chain base-Pkh1/2-Ypk1/2 signaling pathway regulates eisosome assembly and turnover.

    PubMed

    Luo, Guangzuo; Gruhler, Albrecht; Liu, Ying; Jensen, Ole N; Dickson, Robert C

    2008-04-18

    Eisosomes are recently described fungal structures that play roles in the organization of the plasma membrane and endocytosis. Their major protein components are Pil1 and Lsp1, and previous studies showed that these proteins are phosphorylated by the sphingolipid long-chain base-activated Pkh1 and Pkh2 protein kinases in vitro. We show that Pkh1 and Pkh2 phosphorylate Pil1 and Lsp1 in vivo to produce species B, and that heat stress, which activates Pkh1 and Pkh2, generates a more highly phosphorylated species, C. Cells with low Pkh activity lack species B and C and contain abnormally organized eisosomes. To verify that Pil1 phosphorylation is essential for correct eisosome organization, phosphorylated serine and threonine residues were identified and changed to alanines. A variant Pil1 protein lacking five phosphorylation sites did not form eisosomes during log phase growth, indicating that phosphorylation is critical for eisosome organization. We also found that eisosomes are dynamic structures and disassemble when the Ypk protein kinases, which are activated by the sphingolipid-Pkh signaling pathway, are inactivated or when the sphingolipid signal is pharmacologically blocked with myriocin. We conclude that eisosome formation and turnover are regulated by the sphingolipid-Pkh1/2-Ypk1/2 signaling pathway. These data and previous data showing that endocytosis is regulated by the sphingolipid-Pkh1/2-Ypk1/2 signaling pathway suggest that Pkh1 and -2 respond to changes in membrane sphingolipids and transmit this information to eisosomes via Pil1 phosphorylation. Eisosomes then control endocytosis to align the composition and function of the plasma membrane to match demand.

  11. Sphingolipids in the DNA Damage Response

    PubMed Central

    Carroll, Brittany; Donaldson, Cat; Obeid, Lina

    2014-01-01

    Recently, sphingolipid metabolizing enzymes have emerged as important targets of many chemotherapeutics and DNA damaging agents and therefore play significant roles in mediating the physiological response of the cell to DNA damage. In this review we will highlight points of connection between the DNA damage response (DDR) and sphingolipid metabolism; specifically how certain sphingolipid enzymes are regulated in response to DNA damage and how the bioactive lipids produced by these enzymes affect cell fate. PMID:25434743

  12. Counter-regulation of opioid analgesia by glial-derived bioactive sphingolipids

    PubMed Central

    Muscoli, Carolina; Doyle, Tim; Dagostino, Concetta; Bryant, Leesa; Chen, Zhoumou; Watkins, Linda R.; Ryerse, Jan; Bieberich, Erhard; Neumman, William; Salvemini, Daniela

    2010-01-01

    The clinical efficacy of opiates for pain control is severely limited by analgesic tolerance and hyperalgesia. Herein we show that chronic morphine upregulates both the sphingolipid ceramide in spinal astrocytes and microglia, but not neurons, and spinal sphingosine-1-phosphate (S1P), the end-product of ceramide metabolism. Co-administering morphine with intrathecal administration of pharmacological inhibitors of ceramide and S1P blocked formation of spinal S1P and development of hyperalgesia and tolerance in rats. Our results show that spinally formed S1P signals at least in part by 1) modulating glial function, since inhibiting S1P formation blocked increased formation of glial-related pro-inflammatory cytokines, in particular TNF-α, IL-1β α, and IL6, which are known modulators of neuronal excitability, and 2) peroxynitrite-mediated post-translational nitration and inactivation of glial-related enzymes (glutamine synthetase and the glutamate transporter, GLT-1) known to play critical roles in glutamate neurotransmission. Inhibitors of the ceramide metabolic pathway may have therapeutic potential as adjuncts to opiates in relieving suffering from chronic pain. PMID:21084596

  13. Sphingolipids in inflammation: roles and implications.

    PubMed

    Pettus, B J; Chalfant, C E; Hannun, Y A

    2004-06-01

    Sphingolipids, historically described as potential reservoirs for bioactive lipids, presently define a new family of cellular mediators, joining the well-established glycerolipid-derived mediators of signal transduction such as diacylglycerol, phosphatidylinositides, and eicosanoids. Sphingolipid metabolism is clearly involved in the regulation of cell growth, differentiation, and programmed cell death. Indeed, a majority of the greater than four thousand studies conducted on sphingolipids during the past five years were investigations of the role of sphingolipids as cellular bioregulators. Studies spanning more than a decade have shown multiple interactions and intersections of the sphingolipid-mediated pathways and the eicosanoid pathway. This review will discuss the emerging mechanisms by which sphingolipids induce inflammatory responses via the eicosanoid pathway in addition to linking previous literature on sphingolipids and inflammation with newer findings of distinct roles for sphingosine-1-phosphate in regulating cyclooygenase-2 and ceramide-1-phosphate in the regulation of cytosolic phospholipase A2alpha. Finally, the relationship between bioactive sphingolipids and inflammation is discussed.

  14. Sphingolipids inhibit vimentin-dependent cell migration.

    PubMed

    Hyder, Claire L; Kemppainen, Kati; Isoniemi, Kimmo O; Imanishi, Susumu Y; Goto, Hidemasa; Inagaki, Masaki; Fazeli, Elnaz; Eriksson, John E; Törnquist, Kid

    2015-06-01

    The sphingolipids, sphingosine 1-phosphate (S1P) and sphingosylphosphorylcholine (SPC), can induce or inhibit cellular migration. The intermediate filament protein vimentin is an inducer of migration and a marker for epithelial-mesenchymal transition. Given that keratin intermediate filaments are regulated by SPC, with consequences for cell motility, we wanted to determine whether vimentin is also regulated by sphingolipid signalling and whether it is a determinant for sphingolipid-mediated functions. In cancer cells where S1P and SPC inhibited migration, we observed that S1P and SPC induced phosphorylation of vimentin on S71, leading to a corresponding reorganization of vimentin filaments. These effects were sphingolipid-signalling-dependent, because inhibition of either the S1P2 receptor (also known as S1PR2) or its downstream effector Rho-associated kinase (ROCK, for which there are two isoforms ROCK1 and ROCK2) nullified the sphingolipid-induced effects on vimentin organization and S71 phosphorylation. Furthermore, the anti-migratory effect of S1P and SPC could be prevented by expressing S71-phosphorylation-deficient vimentin. In addition, we demonstrated, by using wild-type and vimentin-knockout mouse embryonic fibroblasts, that the sphingolipid-mediated inhibition of migration is dependent on vimentin. These results imply that this newly discovered sphingolipid-vimentin signalling axis exerts brake-and-throttle functions in the regulation of cell migration.

  15. Effects of stereochemistry, saturation, and hydrocarbon chain length on the ability of synthetic constrained azacyclic sphingolipids to trigger nutrient transporter down-regulation, vacuolation, and cell death.

    PubMed

    Perryman, Michael S; Tessier, Jérémie; Wiher, Timothy; O'Donoghue, Heather; McCracken, Alison N; Kim, Seong M; Nguyen, Dean G; Simitian, Grigor S; Viana, Matheus; Rafelski, Susanne; Edinger, Aimee L; Hanessian, Stephen

    2016-09-15

    Constrained analogs containing a 2-hydroxymethylpyrrolidine core of the natural sphingolipids sphingosine, sphinganine, N,N-dimethylsphingosine and N-acetyl variants of sphingosine and sphinganine (C2-ceramide and dihydro-C2-ceramide) were synthesized and evaluated for their ability to down-regulate nutrient transporter proteins and trigger cytoplasmic vacuolation in mammalian cells. In cancer cells, the disruptions in intracellular trafficking produced by these sphingolipids lead to cancer cell death by starvation. Structure activity studies were conducted by varying the length of the hydrocarbon chain, the degree of unsaturation and the presence or absence of an aryl moiety on the appended chains, and stereochemistry at two stereogenic centers. In general, cytotoxicity was positively correlated with nutrient transporter down-regulation and vacuolation. This study was intended to identify structural and functional features in lead compounds that best contribute to potency, and to develop chemical biology tools that could be used to isolate the different protein targets responsible for nutrient transporter loss and cytoplasmic vacuolation. A molecule that produces maximal vacuolation and transporter loss is expected to have the maximal anti-cancer activity and would be a lead compound.

  16. ORM Expression Alters Sphingolipid Homeostasis and Differentially Affects Ceramide Synthase Activity1[OPEN

    PubMed Central

    Kimberlin, Athen N.; Chen, Ming; Dunn, Teresa M.

    2016-01-01

    Sphingolipid synthesis is tightly regulated in eukaryotes. This regulation in plants ensures sufficient sphingolipids to support growth while limiting the accumulation of sphingolipid metabolites that induce programmed cell death. Serine palmitoyltransferase (SPT) catalyzes the first step in sphingolipid biosynthesis and is considered the primary sphingolipid homeostatic regulatory point. In this report, Arabidopsis (Arabidopsis thaliana) putative SPT regulatory proteins, orosomucoid-like proteins AtORM1 and AtORM2, were found to interact physically with Arabidopsis SPT and to suppress SPT activity when coexpressed with Arabidopsis SPT subunits long-chain base1 (LCB1) and LCB2 and the small subunit of SPT in a yeast (Saccharomyces cerevisiae) SPT-deficient mutant. Consistent with a role in SPT suppression, AtORM1 and AtORM2 overexpression lines displayed increased resistance to the programmed cell death-inducing mycotoxin fumonisin B1, with an accompanying reduced accumulation of LCBs and C16 fatty acid-containing ceramides relative to wild-type plants. Conversely, RNA interference (RNAi) suppression lines of AtORM1 and AtORM2 displayed increased sensitivity to fumonisin B1 and an accompanying strong increase in LCBs and C16 fatty acid-containing ceramides relative to wild-type plants. Overexpression lines also were found to have reduced activity of the class I ceramide synthase that uses C16 fatty acid acyl-coenzyme A and dihydroxy LCB substrates but increased activity of class II ceramide synthases that use very-long-chain fatty acyl-coenzyme A and trihydroxy LCB substrates. RNAi suppression lines, in contrast, displayed increased class I ceramide synthase activity but reduced class II ceramide synthase activity. These findings indicate that ORM mediation of SPT activity differentially regulates functionally distinct ceramide synthase activities as part of a broader sphingolipid homeostatic regulatory network. PMID:27506241

  17. Fostering Inflammatory Bowel Disease: Sphingolipid Strategies to Join Forces

    PubMed Central

    Abdel Hadi, Loubna; Di Vito, Clara; Riboni, Laura

    2016-01-01

    Complex sphingolipids are essential structural components of intestinal membranes, providing protection and integrity to the intestinal mucosa and regulating intestinal absorption processes. The role of sphingolipid signaling has been established in numerous cellular events, including intestinal cell survival, growth, differentiation, and apoptosis. A significant body of knowledge demonstrates that intestinal sphingolipids play a crucial role, as such and through their signaling pathways, in immunity and inflammatory disorders. In this review, we report on and discuss the current knowledge on the metabolism, signaling, and functional implications of sphingolipids in inflammatory bowel disease (IBD), focusing on the different aspects of sphingolipid actions on inflammatory responses and on the potential of sphingolipid-targeted molecules as anti-IBD therapeutic agents. PMID:26880864

  18. Fostering Inflammatory Bowel Disease: Sphingolipid Strategies to Join Forces.

    PubMed

    Abdel Hadi, Loubna; Di Vito, Clara; Riboni, Laura

    2016-01-01

    Complex sphingolipids are essential structural components of intestinal membranes, providing protection and integrity to the intestinal mucosa and regulating intestinal absorption processes. The role of sphingolipid signaling has been established in numerous cellular events, including intestinal cell survival, growth, differentiation, and apoptosis. A significant body of knowledge demonstrates that intestinal sphingolipids play a crucial role, as such and through their signaling pathways, in immunity and inflammatory disorders. In this review, we report on and discuss the current knowledge on the metabolism, signaling, and functional implications of sphingolipids in inflammatory bowel disease (IBD), focusing on the different aspects of sphingolipid actions on inflammatory responses and on the potential of sphingolipid-targeted molecules as anti-IBD therapeutic agents.

  19. Interfacial regulation of acid ceramidase activity. Stimulation of ceramide degradation by lysosomal lipids and sphingolipid activator proteins.

    PubMed

    Linke, T; Wilkening, G; Sadeghlar, F; Mozcall, H; Bernardo, K; Schuchman, E; Sandhoff, K

    2001-02-23

    The lysosomal degradation of ceramide is catalyzed by acid ceramidase and requires sphingolipid activator proteins (SAP) as cofactors in vivo. The aim of this study was to investigate how ceramide is hydrolyzed by acid ceramidase at the water-membrane interface in the presence of sphingolipid activator proteins in a liposomal assay system. The degradation of membrane-bound ceramide was significantly increased both in the absence and presence of SAP-D when anionic lysosomal phospholipids such as bis(monoacylglycero)phosphate, phosphatidylinositol, and dolichol phosphate were incorporated into substrate-bearing liposomes. Higher ceramide degradation rates were observed in vesicles with increased membrane curvature. Dilution assays indicated that acid ceramidase remained bound to the liposomal surface during catalysis. Not only SAP-D, but also SAP-C and SAP-A, were found to be stimulators of ceramide hydrolysis in the presence of anionic phospholipids. This finding was confirmed by cell culture studies, in which SAP-A, -C, and -D reduced the amount of ceramide storage observed in fibroblasts of a patient suffering from prosaposin deficiency. Strong protein-lipid interactions were observed for both SAP-D and acid ceramidase in surface plasmon resonance experiments. Maximum binding of SAP-D and acid ceramidase to lipid bilayers occurred at pH 4.0. Our results demonstrate that anionic, lysosomal lipids are required for efficient hydrolysis of ceramide by acid ceramidase.

  20. Inhibition of sphingolipid synthesis improves dyslipidemia in the diet-induced hamster model of insulin resistance: evidence for the role of sphingosine and sphinganine in hepatic VLDL-apoB100 overproduction.

    PubMed

    Dekker, Mark J; Baker, Chris; Naples, Mark; Samsoondar, Josh; Zhang, Rianna; Qiu, Wei; Sacco, Jennifer; Adeli, Khosrow

    2013-05-01

    Sphingolipids have emerged as important bioactive lipid species involved in the pathogenesis of type 2 diabetes and cardiovascular disease. However, little is known of the regulatory role of sphingolipids in dyslipidemia of insulin-resistant states. We employed hamster models of dyslipidemia and insulin resistance to investigate the role of sphingolipids in hepatic VLDL overproduction, induction of insulin resistance, and inflammation. Hamsters were fed either a control chow diet, a high fructose diet, or a diet high in fat, fructose and cholesterol (FFC diet). They were then treated for 2 weeks with vehicle or 0.3 mg/kg myriocin, a potent inhibitor of de novo sphingolipid synthesis. Both fructose and FFC feeding induced significant increases in hepatic sphinganine, which was normalized to chow-fed levels with myriocin (P < 0.05); myriocin also lowered hepatic ceramide content (P < 0.05). Plasma TG and cholesterol as well as VLDL-TG and -apoB100 were similarly reduced with myriocin treatment in all hamsters, regardless of diet. Myriocin treatment also led to improved insulin sensitivity and reduced hepatic SREBP-1c mRNA, though it did not appear to ameliorate the activation of hepatic inflammatory pathways. Importantly, direct treatment of primary hamster hepatocytes ex vivo with C2 ceramide or sphingosine led to an increased secretion of newly synthesized apoB100. Taken together, these data suggest that a) hepatic VLDL-apoB100 overproduction may be stimulated by ceramides and sphingosine and b) inhibition of sphingolipid synthesis can reduce circulating VLDL in hamsters and improve circulating lipids--an effect that is possibly due to improved insulin signaling and reduced lipogenesis but is independent of changes in inflammation.

  1. Selective knockdown of ceramide synthases reveals complex interregulation of sphingolipid metabolism.

    PubMed

    Mullen, Thomas D; Spassieva, Stefka; Jenkins, Russell W; Kitatani, Kazuyuki; Bielawski, Jacek; Hannun, Yusuf A; Obeid, Lina M

    2011-01-01

    Mammalian ceramide synthases 1 to 6 (CerS1-6) generate Cer in an acyl-CoA-dependent manner, and expression of individual CerS has been shown to enhance the synthesis of ceramides with particular acyl chain lengths. However, the contribution of each CerS to steady-state levels of specific Cer species has not been evaluated. We investigated the knockdown of individual CerS in the MCF-7 human breast adenocarcinoma cell line by using small-interfering RNA (siRNA). We found that siRNA-induced downregulation of each CerS resulted in counter-regulation of nontargeted CerS. Additionally, each CerS knockdown produced unique effects on the levels of multiple sphingolipid species. For example, downregulation of CerS2 decreased very long-chain Cer but increased levels of CerS4, CerS5, and CerS6 expression and upregulated long-chain and medium-long-chain sphingolipids. Conversely, CerS6 knockdown decreased C16:0-Cer but increased CerS5 expression and caused non-C16:0 sphingolipids to be upregulated. Knockdown of individual CerS failed to decrease total sphingolipids or upregulate sphingoid bases. Treatment with siRNAs targeting combined CerS, CerS2, CerS5, and CerS6, did not change overall Cer or sphingomyelin mass but caused upregulation of dihydroceramide and hexosyl-ceramide and promoted endoplasmic reticulum stress. These data suggest that sphingolipid metabolism is robustly regulated by both redundancy in CerS-mediated Cer synthesis and counter-regulation of CerS expression.

  2. Monogenic neurological disorders of sphingolipid metabolism.

    PubMed

    Sabourdy, Frédérique; Astudillo, Leonardo; Colacios, Céline; Dubot, Patricia; Mrad, Marguerite; Ségui, Bruno; Andrieu-Abadie, Nathalie; Levade, Thierry

    2015-08-01

    Sphingolipids comprise a wide variety of molecules containing a sphingoid long-chain base that can be N-acylated. These lipids are particularly abundant in the central nervous system, being membrane components of neurons as well as non-neuronal cells. Direct evidence that these brain lipids play critical functions in brain physiology is illustrated by the dramatic consequences of genetic disturbances of their metabolism. Inherited defects of both synthesis and catabolism of sphingolipids are now identified in humans. These monogenic disorders are due to mutations in the genes encoding for the enzymes that catalyze either the formation or degradation of simple sphingolipids such as ceramides, or complex sphingolipids like glycolipids. They cause varying degrees of central nervous system dysfunction, quite similarly to the neurological disorders induced in mice by gene disruption of the corresponding enzymes. Herein, the enzyme deficiencies and metabolic alterations that underlie these diseases are reviewed. Their possible pathophysiological mechanisms and the functions played by sphingolipids one can deduce from these conditions are discussed. This article is part of a Special Issue entitled Brain Lipids.

  3. Metabolism, Physiological Role, and Clinical Implications of Sphingolipids in Gastrointestinal Tract

    PubMed Central

    Łukaszuk, Bartłomiej; Piotrowska, Dominika M.; Wiesiołek, Patrycja; Chabowska, Anna Małgorzata; Żendzian-Piotrowska, Małgorzata

    2013-01-01

    Sphingolipids in digestive system are responsible for numerous important physiological and pathological processes. In the membrane of gut epithelial cells, sphingolipids provide structural integrity, regulate absorption of some nutrients, and act as receptors for many microbial antigens and their toxins. Moreover, bioactive sphingolipids such as ceramide or sphingosine-1-phosphate regulate cellular growth, differentiation, and programmed cell death—apoptosis. Although it is well established that sphingolipids have clinical implications in gastrointestinal tumorigenesis or inflammation, further studies are needed to fully explore the role of sphingolipids in neoplastic and inflammatory diseases in gastrointestinal tract. Pharmacological agents which regulate metabolism of sphingolipids can be potentially used in the management of colorectal cancer or inflammatory bowel diseases. The aim of this work is to critically the review physiological and pathological roles of sphingolipids in the gastrointestinal tract. PMID:24083248

  4. Viral trans-dominant manipulation of algal sphingolipids.

    PubMed

    Michaelson, Louise V; Dunn, Teresa M; Napier, Johnathan A

    2010-12-01

    Emiliania huxleyi is the host for the coccolithovirus (EhV), which is responsible for the demise of large oceanic blooms formed by this alga. The EhV-86 virus genome sequence has identified several genes apparently involved in sphingolipid metabolism. Recently, an unusual glucosylceramide from E. huxleyi infected with EhV-86 was isolated, implicating sphingolipids in the lysis of this alga. However, the EhV-86-encoded genes contain only a subset of the activities required to generate the novel sphingolipid, implying that its synthesis is the result of coordinated interactions between algal- and viral-encoded biosynthetic enzymes. Here, we discuss the likely role for EhV-86 open reading frames (ORFs) in the synthesis of novel sphingolipids and also consider the concept of the trans-dominant manipulation of lipid metabolism.

  5. Drug targeting of sphingolipid metabolism: sphingomyelinases and ceramidases

    PubMed Central

    Canals, Daniel; Perry, David M; Jenkins, Russell W; Hannun, Yusuf A

    2011-01-01

    Sphingolipids represent a class of diverse bioactive lipid molecules that are increasingly appreciated as key modulators of diverse physiologic and pathophysiologic processes that include cell growth, cell death, autophagy, angiogenesis, and stress and inflammatory responses. Sphingomyelinases and ceramidases are key enzymes of sphingolipid metabolism that regulate the formation and degradation of ceramide, one of the most intensely studied classes of sphingolipids. Improved understanding of these enzymes that control not only the levels of ceramide but also the complex interconversion of sphingolipid metabolites has provided the foundation for the functional analysis of the roles of sphingolipids. Our current understanding of the roles of various sphingolipids in the regulation of different cellular processes has come from loss-of-function/gain-of-function studies utilizing genetic deletion/downregulation/overexpression of enzymes of sphingolipid metabolism (e.g. knockout animals, RNA interference) and from the use of pharmacologic inhibitors of these same enzymes. While genetic approaches to evaluate the functional roles of sphingolipid enzymes have been instrumental in advancing the field, the use of pharmacologic inhibitors has been equally important in identifying new roles for sphingolipids in important cellular processes.The latter also promises the development of novel therapeutic targets with implications for cancer therapy, inflammation, diabetes, and neurodegeneration. In this review, we focus on the status and use of pharmacologic compounds that inhibit sphingomyelinases and ceramidases, and we will review the history, current uses and future directions for various small molecule inhibitors, and will highlight studies in which inhibitors of sphingolipid metabolizing enzymes have been used to effectively treat models of human disease. PMID:21615386

  6. Sphingolipid Metabolism, Oxidant Signaling, and Contractile Function of Skeletal Muscle

    PubMed Central

    Nikolova-Karakashian, Mariana N.

    2011-01-01

    Abstract Significance Sphingolipids are a class of bioactive lipids that regulate diverse cell functions. Ceramide, sphingosine, and sphingosine-1-phosphate accumulate in tissues such as liver, brain, and lung under conditions of cellular stress, including oxidative stress. The activity of some sphingolipid metabolizing enzymes, chiefly the sphingomyelinases, is stimulated during inflammation and in response to oxidative stress. Ceramide, the sphingomyelinase product, as well as the ceramide metabolite, sphingosine-1-phosphate, can induce the generation of more reactive oxygen species, propagating further inflammation. Recent Advances This review article summarizes information on sphingolipid biochemistry and signaling pertinent to skeletal muscle and describes the potential influence of sphingolipids on contractile function. Critical Issues It encompasses topics related to (1) the pathways for complex sphingolipid biosynthesis and degradation, emphasizing sphingolipid regulation in various muscle fiber types and subcellular compartments; (2) the emerging evidence that implicates ceramide, sphingosine, and sphingosine-1-phosphate as regulators of muscle oxidant activity, and (3) sphingolipid effects on contractile function and fatigue. Future Directions We propose that prolonged inflammatory conditions alter ceramide, sphingosine, and sphingosine-1-phosphate levels in skeletal muscle and that these changes promote the weakness, premature fatigue, and cachexia that plague individuals with heart failure, cancer, diabetes, and other chronic inflammatory diseases. Antioxid. Redox Signal. 15, 2501–2517. PMID:21453197

  7. Harnessing the power of yeast to elucidate the role of sphingolipids in metabolic and signaling processes pertinent to psychiatric disorders

    PubMed Central

    Jadhav, Shyamalagauri; Greenberg, Miriam L

    2015-01-01

    The development of therapies for neuropsychiatric disorders is hampered by the lack of understanding of the mechanisms underlying their pathologies. While aberrant sphingolipid metabolism is associated with psychiatric illness, the role of sphingolipids in these disorders is not understood. The genetically tractable yeast model can be exploited in order to elucidate the cellular consequences of sphingolipid perturbation. Hypotheses generated from studies in yeast and tested in mammalian cells may contribute to our understanding of the role of sphingolipids in psychiatric disorders and to the development of new treatments. Here, we compare sphingolipid metabolism in yeast and mammalian cells, discuss studies implicating sphingolipids in psychiatric disorders and propose approaches that utilize yeast in order to elucidate sphingolipid function and identify drugs that target sphingolipid synthesis. PMID:25750665

  8. Novel drugs targeting sphingolipid metabolism.

    PubMed

    Bhabak, Krishna P; Arenz, Christoph

    2013-01-01

    While the evidence for an involvement of sphingolipids (SLs) in a variety of diseases is rapidly increasing, the development of sphingolipid-related drugs is still in its infancy. In fact, the recently FDA-approved fingolimod or FTY-720 (see chapter by J. Pfeilschifter for more information) is the first drug on the market to interfere with sphingolipid signaling. The reasons for this lagging are manifold and within this chapter we try to name some of them. Ceramide is in the center of sphingolipid metabolism. We describe the most important and most recent inhibitors for enzymes controlling cellular ceramide levels.

  9. Role of Sphingolipids in the Pathobiology of Lung Inflammation

    PubMed Central

    Ghidoni, Riccardo; Caretti, Anna; Signorelli, Paola

    2015-01-01

    Sphingolipid bioactivities in the respiratory airways and the roles of the proteins that handle them have been extensively investigated. Gas or inhaled particles or microorganisms come into contact with mucus components, epithelial cells, blood barrier, and immune surveillance within the airways. Lung structure and functionality rely on a complex interplay of polar and hydrophobic structures forming the surfactant layer and governing external-internal exchanges, such as glycerol-phospholipids sphingolipids and proteins. Sphingolipids act as important signaling mediators involved in the control of cell survival and stress response, as well as secreted molecules endowed with inflammation-regulatory activities. Most successful respiratory infection and injuries evolve in the alveolar compartment, the critical lung functional unit involved in gas exchange. Sphingolipid altered metabolism in this compartment is closely related to inflammatory reaction and ceramide increase, in particular, favors the switch to pathological hyperinflammation. This short review explores a few mechanisms underlying sphingolipid involvement in the healthy lung (surfactant production and endothelial barrier maintenance) and in a selection of lung pathologies in which the impact of sphingolipid synthesis and metabolism is most apparent, such as acute lung injury, or chronic pathologies such as cystic fibrosis and chronic obstructive pulmonary disease. PMID:26770018

  10. The Yeast Sphingolipid Signaling Landscape

    PubMed Central

    Montefusco, David J.; Matmati, Nabil

    2014-01-01

    Sphingolipids are recognized as signaling mediators in a growing number of pathways, and represent potential targets to address many diseases. The study of sphingolipid signaling in yeast has created a number of breakthroughs in the field, and has the potential to lead future advances. The aim of this article is to provide an inclusive view of two major frontiers in yeast sphingolipid signaling. In the first section, several key studies in the field of sphingolipidomics are consolidated to create a yeast sphingolipidome that ranks nearly all known sphingolipid species by their level in a resting yeast cell. The second section presents an overview of most known phenotypes identified for sphingolipid gene mutants, presented with the intention of illuminating not yet discovered connections outside and inside of the field. PMID:24220500

  11. Involvement of Sphingolipids in Ethanol Neurotoxicity in the Developing Brain

    PubMed Central

    Saito, Mariko; Saito, Mitsuo

    2013-01-01

    Ethanol-induced neuronal death during a sensitive period of brain development is considered one of the significant causes of fetal alcohol spectrum disorders (FASD). In rodent models, ethanol triggers robust apoptotic neurodegeneration during a period of active synaptogenesis that occurs around the first two postnatal weeks, equivalent to the third trimester in human fetuses. The ethanol-induced apoptosis is mitochondria-dependent, involving Bax and caspase-3 activation. Such apoptotic pathways are often mediated by sphingolipids, a class of bioactive lipids ubiquitously present in eukaryotic cellular membranes. While the central role of lipids in ethanol liver toxicity is well recognized, the involvement of sphingolipids in ethanol neurotoxicity is less explored despite mounting evidence of their importance in neuronal apoptosis. Nevertheless, recent studies indicate that ethanol-induced neuronal apoptosis in animal models of FASD is mediated or regulated by cellular sphingolipids, including via the pro-apoptotic action of ceramide and through the neuroprotective action of GM1 ganglioside. Such sphingolipid involvement in ethanol neurotoxicity in the developing brain may provide unique targets for therapeutic applications against FASD. Here we summarize findings describing the involvement of sphingolipids in ethanol-induced apoptosis and discuss the possibility that the combined action of various sphingolipids in mitochondria may control neuronal cell fate. PMID:24961420

  12. Involvement of sphingolipids in ethanol neurotoxicity in the developing brain.

    PubMed

    Saito, Mariko; Saito, Mitsuo

    2013-04-26

    Ethanol-induced neuronal death during a sensitive period of brain development is considered one of the significant causes of fetal alcohol spectrum disorders (FASD). In rodent models, ethanol triggers robust apoptotic neurodegeneration during a period of active synaptogenesis that occurs around the first two postnatal weeks, equivalent to the third trimester in human fetuses. The ethanol-induced apoptosis is mitochondria-dependent, involving Bax and caspase-3 activation. Such apoptotic pathways are often mediated by sphingolipids, a class of bioactive lipids ubiquitously present in eukaryotic cellular membranes. While the central role of lipids in ethanol liver toxicity is well recognized, the involvement of sphingolipids in ethanol neurotoxicity is less explored despite mounting evidence of their importance in neuronal apoptosis. Nevertheless, recent studies indicate that ethanol-induced neuronal apoptosis in animal models of FASD is mediated or regulated by cellular sphingolipids, including via the pro-apoptotic action of ceramide and through the neuroprotective action of GM1 ganglioside. Such sphingolipid involvement in ethanol neurotoxicity in the developing brain may provide unique targets for therapeutic applications against FASD. Here we summarize findings describing the involvement of sphingolipids in ethanol-induced apoptosis and discuss the possibility that the combined action of various sphingolipids in mitochondria may control neuronal cell fate.

  13. Phospholipid and sphingolipid metabolism in Leishmania

    PubMed Central

    Zhang, Kai; Beverley, Stephen M.

    2009-01-01

    In many eukaryotes, phospholipids (PLs) and sphingolipids (SLs) are abundant membrane components and reservoirs for important signaling molecules. In Leishmania, the composition, metabolism, and function of PLs and SLs differ significantly from those in mammalian cells. Although only a handful of enzymes have been experimentally characterized, available data suggest many steps of PL/SL metabolism are critical for Leishmania viability and/or virulence, and could be a source for new drug targets. Further studies of genes involved in the synthesis (de novo and salvage) and degradation of PLs and SLs will reveal their diverse effects on Leishmania pathogenesis. PMID:20026359

  14. A systematic simulation of the effect of salicylic acid on sphingolipid metabolism

    PubMed Central

    Shi, Chao; Yin, Jian; Liu, Zhe; Wu, Jian-Xin; Zhao, Qi; Ren, Jian; Yao, Nan

    2015-01-01

    The phytohormone salicylic acid (SA) affects plant development and defense responses. Recent studies revealed that SA also participates in the regulation of sphingolipid metabolism, but the details of this regulation remain to beexplored. Here, we use in silico Flux Balance Analysis (FBA) with published microarray data to construct a whole-cell simulation model, including 23 pathways, 259 reactions, and 172 metabolites, to predict the alterations in flux of major sphingolipid species after treatment with exogenous SA. This model predicts significant changes in fluxes of certain sphingolipid species after SA treatment, changes that likely trigger downstream physiological and phenotypic effects. To validate the simulation, we used 15N-labeled metabolic turnover analysis to measure sphingolipid contents and turnover rate in Arabidopsis thaliana seedlings treated with SA or the SA analog benzothiadiazole (BTH). The results show that both SA and BTH affect sphingolipid metabolism, altering the concentrations of certain species and also changing the optimal flux distribution and turnover rate of sphingolipids. Our strategy allows us to estimate sphingolipid fluxes on a short time scale and gives us a systemic view of the effect of SA on sphingolipid homeostasis. PMID:25859253

  15. Membrane engineering of S. cerevisiae targeting sphingolipid metabolism

    PubMed Central

    Lindahl, Lina; Santos, Aline X. S.; Olsson, Helén; Olsson, Lisbeth; Bettiga, Maurizio

    2017-01-01

    The sustainable production of fuels and chemicals using microbial cell factories is now well established. However, many microbial production processes are still limited in scale due to inhibition from compounds that are present in the feedstock or are produced during fermentation. Some of these inhibitors interfere with cellular membranes and change the physicochemical properties of the membranes. Another group of molecules is dependent on their permeation rate through the membrane for their inhibition. We have investigated the use of membrane engineering to counteract the negative effects of inhibitors on the microorganism with focus on modulating the abundance of complex sphingolipids in the cell membrane of Saccharomyces cerevisiae. Overexpression of ELO3, involved in fatty acid elongation, and AUR1, which catalyses the formation of complex sphingolipids, had no effect on the membrane lipid profile or on cellular physiology. Deletion of the genes ORM1 and ORM2, encoding negative regulators of sphingolipid biosynthesis, decreased cell viability and considerably reduced phosphatidylinositol and complex sphingolipids. Additionally, combining ELO3 and AUR1 overexpression with orm1/2Δ improved cell viability and increased fatty acyl chain length compared with only orm1/2Δ. These findings can be used to further study the sphingolipid metabolism, as well as giving guidance in membrane engineering. PMID:28145511

  16. Tumor PDT-associated immune response: relevance of sphingolipids

    NASA Astrophysics Data System (ADS)

    Korbelik, Mladen; Merchant, Soroush; Separovic, Duska M.

    2010-02-01

    Sphingolipids have become recognized as essential effector molecules in signal transduction with involvement in various aspects of cell function and death, immune response and cancer treatment response. Major representatives of sphingolipids family, ceramide, sphingosine and sphingosine-1-phosphate (S1P), have attracted interest in their relevance to tumor response to photodynamic therapy (PDT) because of their roles as enhancers of apoptosis, mediators of cell growth and vasculogenesis, and regulators of immune response. Our recent in vivo studies with mouse tumor models have confirmed that PDT treatment has a pronounced impact on sphingolipid profile in the targeted tumor and that significant advances in therapeutic gain with PDT can be attained by combining this modality with adjuvant treatment with ceramide analog LCL29.

  17. Rice Stripe Virus Infection Alters mRNA Levels of Sphingolipid-Metabolizing Enzymes and Sphingolipids Content in Laodelphax striatellus

    PubMed Central

    Li, Fei-Qiang; Bai, Yue-Liang; Shi, Xiao-Xiao; Zhu, Mu-Fei; Zhang, Min-Jing; Mao, Cun-Gui; Zhu, Zeng-Rong

    2017-01-01

    Sphingolipids and their metabolites have been implicated in viral infection and replication in mammal cells but how their metabolizing enzymes in the host are regulated by viruses remains largely unknown. Here we report the identification of 12 sphingolipid genes and their regulation by Rice stripe virus in the small brown planthopper (Laodelphax striatellus Fallén), a serious pest of rice throughout eastern Asia. According to protein sequence similarity, we identified 12 sphingolipid enzyme genes in L. striatellus. By comparing their mRNA levels in viruliferous versus nonviruliferous L. striatellus at different life stages by qPCR, we found that RSV infection upregulated six genes (LsCGT1, LsNAGA1, LsSGPP, LsSMPD4, LsSMS, and LsSPT) in most stages of L. striatellus. Especially, four genes (LsCGT1, LsSMPD2, LsNAGA1, and LsSMS) and another three genes (LsNAGA1, LsSGPP, and LsSMS) were significantly upregulated in viruliferous third-instar and fourth-instar nymphs, respectively. HPLC-MS/MS results showed that RSV infection increased the levels of various ceramides, such as Cer18:0, Cer20:0, and Cer22:0 species, in third and fourth instar L. striatellus nymphs. Together, these results demonstrate that RSV infection alters the transcript levels of various sphingolipid enzymes and the contents of sphingolipids in L. striatellus, indicating that sphingolipids may be important for RSV infection or replication in L. striatellus. PMID:28130458

  18. Bile acids: regulation of synthesis.

    PubMed

    Chiang, John Y L

    2009-10-01

    Bile acids are physiological detergents that generate bile flow and facilitate intestinal absorption and transport of lipids, nutrients, and vitamins. Bile acids also are signaling molecules and inflammatory agents that rapidly activate nuclear receptors and cell signaling pathways that regulate lipid, glucose, and energy metabolism. The enterohepatic circulation of bile acids exerts important physiological functions not only in feedback inhibition of bile acid synthesis but also in control of whole-body lipid homeostasis. In the liver, bile acids activate a nuclear receptor, farnesoid X receptor (FXR), that induces an atypical nuclear receptor small heterodimer partner, which subsequently inhibits nuclear receptors, liver-related homolog-1, and hepatocyte nuclear factor 4alpha and results in inhibiting transcription of the critical regulatory gene in bile acid synthesis, cholesterol 7alpha-hydroxylase (CYP7A1). In the intestine, FXR induces an intestinal hormone, fibroblast growth factor 15 (FGF15; or FGF19 in human), which activates hepatic FGF receptor 4 (FGFR4) signaling to inhibit bile acid synthesis. However, the mechanism by which FXR/FGF19/FGFR4 signaling inhibits CYP7A1 remains unknown. Bile acids are able to induce FGF19 in human hepatocytes, and the FGF19 autocrine pathway may exist in the human livers. Bile acids and bile acid receptors are therapeutic targets for development of drugs for treatment of cholestatic liver diseases, fatty liver diseases, diabetes, obesity, and metabolic syndrome.

  19. Brave little yeast, please guide us to thebes: sphingolipid function in S. cerevisiae.

    PubMed

    Schneiter, R

    1999-12-01

    Sphingolipids typically cover the exoplasmic leaflet of the plasma membrane of eukaryotic cells. They differ from the more abundant glycerophospholipids in that they contain ceramide instead of diacylglycerol as a hydrophobic anchor. Why did nature choose to invent this complex class of lipids, and why do eukaryotic cells follow elaborate remodelling pathways in order to generate dozens to hundreds of different molecular species of sphingolipid, depending on cell type? Yeast may, once again, serve as a model to dissect sphingolipid function at various levels. Almost the complete pathway for sphingolipid synthesis in yeast has been uncovered during the past two decades. More recently, key enzymes in sphingolipid degradation and signalling have been identified. Together with a wealth of genetic data obtained from the characterization of various suppressor mutants, this information now allows for an unprecedented analysis of sphingolipid function in this organism. This overview summarizes recent data on sphingolipid function in cell signalling, their role in the heat-stress response and Ca(2+) homeostasis, and addresses their function in transport of glycosylphosphatidylinositol-anchored proteins.

  20. Exploring the role of sphingolipid machinery during the epithelial to mesenchymal transition program using an integrative approach

    PubMed Central

    Meshcheryakova, Anastasia; Köfeler, Harald C.; Triebl, Alexander; Mungenast, Felicitas; Heinze, Georg; Gerner, Christopher; Zimmermann, Philip; Jaritz, Markus; Mechtcheriakova, Diana

    2016-01-01

    The epithelial to mesenchymal transition (EMT) program is activated in epithelial cancer cells and facilitates their ability to metastasize based on enhanced migratory, proliferative, anti-apoptotic, and pluripotent capacities. Given the fundamental impact of sphingolipid machinery to each individual process, the sphingolipid-related mechanisms might be considered among the most prominent drivers/players of EMT; yet, there is still limited knowledge. Given the complexity of the interconnected sphingolipid system, which includes distinct sphingolipid mediators, their synthesizing enzymes, receptors and transporters, we herein apply an integrative approach for assessment of the sphingolipid-associated mechanisms underlying EMT program. We created the sphingolipid-/EMT-relevant 41-gene/23-gene signatures which were applied to denote transcriptional events in a lung cancer cell-based EMT model. Based on defined 35-gene sphingolipid/EMT-attributed signature of regulated genes, we show close associations between EMT markers, genes comprising the sphingolipid network at multiple levels and encoding sphingosine 1-phosphate (S1P)-/ceramide-metabolizing enzymes, S1P and lysophosphatidic acid (LPA) receptors and S1P transporters, pluripotency genes and inflammation-related molecules, and demonstrate the underlying biological pathways and regulators. Mass spectrometry-based sphingolipid analysis revealed an EMT-attributed shift towards increased S1P and LPA accompanied by reduced ceramide levels. Notably, using transcriptomics data across various cell-based perturbations and neoplastic tissues (24193 arrays), we identified the sphingolipid/EMT signature primarily in lung adenocarcinoma tissues; besides, bladder, colorectal and prostate cancers were among the top-ranked. The findings also highlight novel regulatory associations between influenza virus and the sphingolipid/EMT-associated mechanisms. In sum, data propose the multidimensional contribution of sphingolipid machinery

  1. Nonrenal regulation of EPO synthesis.

    PubMed

    Weidemann, Alexander; Johnson, Randall S

    2009-04-01

    Erythropoietin (EPO) is a circulating glycoprotein hormone whose principal function is thought to be red blood cell production. It is a classic example of a hypoxia-inducible gene, and studies of the induction of EPO synthesis by low oxygen led to the discovery of a widespread system of hypoxia-inducible transcription factors. Tissue-specific expression of the EPO gene is tightly controlled, and in the adult organism the kidney produces around 90% of systemic EPO. Before birth, the liver is the main site of EPO production; factors contributing to the liver-to-kidney switch are still elusive, but may provide clues to the tissue-specificity of EPO gene expression. EPO has also been detected in non-erythropoietic tissues such as the brain, where it is suggested to exert local protective effects. Apart from classical ways of regulating renal EPO during hypoxia and anemia, novel pathways have been discovered that demonstrate that other organ systems in the adult might not only be important for the production of EPO but also for modulating the hypoxic EPO response. Knowledge of the molecular bases of these non-renal pathways will eventually help to develop pharmacological strategies to induce endogenous EPO production when the main source, the kidney, is significantly impaired. This review will provide an overview of the molecular aspects of EPO gene regulation by hypoxia-inducible transcription factors and of the tissue-specific regulation of EPO production in adult mammals. Insights into the biology of EPO production in genetically modified animals, with an emphasis on recent advances in the understanding of non-renal EPO regulation, will be discussed.

  2. CFTR and sphingolipids mediate hypoxic pulmonary vasoconstriction

    PubMed Central

    Tabeling, Christoph; Yu, Hanpo; Wang, Liming; Ranke, Hannes; Goldenberg, Neil M.; Zabini, Diana; Noe, Elena; Krauszman, Adrienn; Gutbier, Birgitt; Yin, Jun; Schaefer, Michael; Arenz, Christoph; Hocke, Andreas C.; Suttorp, Norbert; Proia, Richard L.; Witzenrath, Martin; Kuebler, Wolfgang M.

    2015-01-01

    Hypoxic pulmonary vasoconstriction (HPV) optimizes pulmonary ventilation-perfusion matching in regional hypoxia, but promotes pulmonary hypertension in global hypoxia. Ventilation-perfusion mismatch is a major cause of hypoxemia in cystic fibrosis. We hypothesized that cystic fibrosis transmembrane conductance regulator (CFTR) may be critical in HPV, potentially by modulating the response to sphingolipids as mediators of HPV. HPV and ventilation-perfusion mismatch were analyzed in isolated mouse lungs or in vivo. Ca2+ mobilization and transient receptor potential canonical 6 (TRPC6) translocation were studied in human pulmonary (PASMCs) or coronary (CASMCs) artery smooth muscle cells. CFTR inhibition or deficiency diminished HPV and aggravated ventilation-perfusion mismatch. In PASMCs, hypoxia caused CFTR to interact with TRPC6, whereas CFTR inhibition attenuated hypoxia-induced TRPC6 translocation to caveolae and Ca2+ mobilization. Ca2+ mobilization by sphingosine-1-phosphate (S1P) was also attenuated by CFTR inhibition in PASMCs, but amplified in CASMCs. Inhibition of neutral sphingomyelinase (nSMase) blocked HPV, whereas exogenous nSMase caused TRPC6 translocation and vasoconstriction that were blocked by CFTR inhibition. nSMase- and hypoxia-induced vasoconstriction, yet not TRPC6 translocation, were blocked by inhibition or deficiency of sphingosine kinase 1 (SphK1) or antagonism of S1P receptors 2 and 4 (S1P2/4). S1P and nSMase had synergistic effects on pulmonary vasoconstriction that involved TRPC6, phospholipase C, and rho kinase. Our findings demonstrate a central role of CFTR and sphingolipids in HPV. Upon hypoxia, nSMase triggers TRPC6 translocation, which requires its interaction with CFTR. Concomitant SphK1-dependent formation of S1P and activation of S1P2/4 result in phospholipase C-mediated TRPC6 and rho kinase activation, which conjointly trigger vasoconstriction. PMID:25829545

  3. Separation and identification of major plant sphingolipid classes from leaves.

    PubMed

    Markham, Jonathan E; Li, Jia; Cahoon, Edgar B; Jaworski, Jan G

    2006-08-11

    Sphingolipids are major components of the plasma membrane, tonoplast, and other endomembranes of plant cells. Previous compositional analyses have focused only on individual sphingolipid classes because of the widely differing polarities of plant sphingolipids. Consequently, the total content of sphingolipid classes in plants has yet to be quantified. In addition, the major polar sphingolipid class in the model plant Arabidopsis thaliana has not been previously determined. In this report, we describe the separation and quantification of sphingolipid classes from A. thaliana leaves using hydrolysis of sphingolipids and high performance liquid chromatography (HPLC) analysis of o-phthaldialdehyde derivatives of the released long-chain bases to monitor the separation steps. An extraction solvent that contained substantial proportions of water was used to solubilized >95% of the sphingolipids from leaves. Neutral and charged sphingolipids were then partitioned by anion exchange solid phase extraction. HPLC analysis of the charged lipid fraction from A. thaliana revealed only one major anionic sphingolipid class, which was identified by mass spectrometry as hexose-hexuronic-inositolphosphoceramide. The neutral sphingolipids were predominantly composed of monohexosylceramide with lesser amounts of ceramides. Extraction and separation of sphingolipids from soybean and tomato showed that, like A. thaliana, the neutral sphingolipids consisted of ceramide and monohexosylceramides; however, the major polar sphingolipid was found to be N-acetyl-hexosamine-hexuronic-inositolphosphoceramide. In extracts from A. thaliana leaves, hexosehexuronic-inositolphosphoceramides, monohexosylceramides, and ceramides accounted for approximately 64, 34, and 2% of the total sphingolipids, respectively, suggesting an important role for the anionic sphingolipids in plant membranes.

  4. Effect of sterol carrier protein-2 expression on sphingolipid distribution in plasma membrane lipid rafts/caveolae.

    PubMed

    Atshaves, Barbara P; Jefferson, John R; McIntosh, Avery L; Gallegos, Adalberto; McCann, Bonnie M; Landrock, Kerstin K; Kier, Ann B; Schroeder, Friedhelm

    2007-10-01

    Although sphingolipids are highly important signaling molecules enriched in lipid rafts/caveolae, relatively little is known regarding factors such as sphingolipid binding proteins that may regulate the distribution of sphingolipids to lipid rafts/caveolae of living cells. Since early work demonstrated that sterol carrier protein-2 (SCP-2) enhanced glycosphingolipid transfer from membranes in vitro, the effect of SCP-2 expression on sphingolipid distribution to lipid rafts/caveolae in living cells was examined. Using a non-detergent affinity chromatography method to isolate lipid rafts/caveolae and non-rafts from purified L-cell plasma membranes, it was shown that lipid rafts/caveolae were highly enriched in multiple sphingolipid species including ceramides, acidic glycosphingolipids (ganglioside GM1); neutral glycosphingolipids (monohexosides, dihexosides, globosides), and sphingomyelin as compared to non-raft domains. SCP-2 overexpression further enriched the content of total sphingolipids and select sphingolipid species in the lipid rafts/caveolae domains. Analysis of fluorescence binding and displacement data revealed that purified human recombinant SCP-2 exhibited high binding affinity (nanomolar range) for all sphingolipid classes tested. The binding affinity decreased in the following order: ceramides > acidic glycosphingolipid (ganglioside GM1) > neutral glycosphingolipid (monohexosides, hexosides, globosides) > sphingomyelin. Enrichment of individual sphingolipid classes to lipid rafts/caveolae versus non-rafts in SCP-2 expressing plasma membranes followed closely with those classes most strongly bound to SCP-2 (ceramides, GM1 > the neutral glycosphingolipids (monohexosides, dihexosides, and globosides) > sphingomyelin). Taken together these data suggested that SCP-2 acts to selectively regulate sphingolipid distribution to lipid rafts/caveolae in living cells.

  5. Sphingolipid and Ceramide Homeostasis: Potential Therapeutic Targets

    PubMed Central

    Young, Simon A.; Mina, John G.; Denny, Paul W.; Smith, Terry K.

    2012-01-01

    Sphingolipids are ubiquitous in eukaryotic cells where they have been attributed a plethora of functions from the formation of structural domains to polarized cellular trafficking and signal transduction. Recent research has identified and characterised many of the key enzymes involved in sphingolipid metabolism and this has led to a heightened interest in the possibility of targeting these processes for therapies against cancers, Alzheimer's disease, and numerous important human pathogens. In this paper we outline the major pathways in eukaryotic sphingolipid metabolism and discuss these in relation to disease and therapy for both chronic and infectious conditions. PMID:22400113

  6. Symbiont-derived sphingolipids modulate mucosal homeostasis and B cells in teleost fish.

    PubMed

    Sepahi, Ali; Cordero, Héctor; Goldfine, Howard; Esteban, Maria Ángeles; Salinas, Irene

    2016-12-14

    Symbiotic bacteria and mucosal immunoglobulins have co-evolved for millions of years in vertebrate animals. Symbiotic bacteria products are known to modulate different aspects of the host immune system. We recently reported that Flectobacillus major is a predominant species that lives in the gill and skin mucosal surfaces of rainbow trout (Oncorhynchus mykiss). F. major is known to produce sphingolipids of a unique molecular structure. Here we propose a role for F. major and its sphingolipids in the regulation of B cell populations in rainbow trout, as well as an essential role for sphingolipids in trout mucosal homeostasis. We found that F. major-specific IgT titers are confined to the gill and skin mucus, whereas F. major-specific IgM titers are only detected in serum. Live F. major cells are able to stimulate sustained IgT expression and secretion in gills. F. major sphingolipids modulate the growth of trout total skin and gill symbiotic bacteria. In vivo systemic administration of F. major sphingolipids changes the proportion of IgT(+) to IgM(+) B cells in trout HK. These results demonstrate the key role of the symbiont F. major and its sphingolipids in mucosal homeostasis via the modulation of mucosal and systemic Igs and B cells.

  7. Symbiont-derived sphingolipids modulate mucosal homeostasis and B cells in teleost fish

    PubMed Central

    Sepahi, Ali; Cordero, Héctor; Goldfine, Howard; Esteban, Maria Ángeles; Salinas, Irene

    2016-01-01

    Symbiotic bacteria and mucosal immunoglobulins have co-evolved for millions of years in vertebrate animals. Symbiotic bacteria products are known to modulate different aspects of the host immune system. We recently reported that Flectobacillus major is a predominant species that lives in the gill and skin mucosal surfaces of rainbow trout (Oncorhynchus mykiss). F. major is known to produce sphingolipids of a unique molecular structure. Here we propose a role for F. major and its sphingolipids in the regulation of B cell populations in rainbow trout, as well as an essential role for sphingolipids in trout mucosal homeostasis. We found that F. major-specific IgT titers are confined to the gill and skin mucus, whereas F. major-specific IgM titers are only detected in serum. Live F. major cells are able to stimulate sustained IgT expression and secretion in gills. F. major sphingolipids modulate the growth of trout total skin and gill symbiotic bacteria. In vivo systemic administration of F. major sphingolipids changes the proportion of IgT+ to IgM+ B cells in trout HK. These results demonstrate the key role of the symbiont F. major and its sphingolipids in mucosal homeostasis via the modulation of mucosal and systemic Igs and B cells. PMID:27966609

  8. Disruption of sphingolipid metabolism augments ceramide-induced autophagy in preeclampsia.

    PubMed

    Melland-Smith, Megan; Ermini, Leonardo; Chauvin, Sarah; Craig-Barnes, Hayley; Tagliaferro, Andrea; Todros, Tullia; Post, Martin; Caniggia, Isabella

    2015-04-03

    Bioactive sphingolipids including ceramides are involved in a variety of pathophysiological processes by regulating cell death and survival. The objective of the current study was to examine ceramide metabolism in preeclampsia, a serious disorder of pregnancy characterized by oxidative stress, and increased trophoblast cell death and autophagy. Maternal circulating and placental ceramide levels quantified by tandem mass spectrometry were elevated in pregnancies complicated by preeclampsia. Placental ceramides were elevated due to greater de novo synthesis via high serine palmitoyltransferase activity and reduced lysosomal breakdown via diminished ASAH1 expression caused by TGFB3-induced E2F4 transcriptional repression. SMPD1 activity was reduced; hence, sphingomyelin degradation by SMPD1 did not contribute to elevated ceramide levels in preeclampsia. Oxidative stress triggered similar changes in ceramide levels and acid hydrolase expression in villous explants and trophoblast cells. MALDI-imaging mass spectrometry localized the ceramide increases to the trophophoblast layers and syncytial knots of placentae from pregnancies complicated by preeclampsia. ASAH1 inhibition or ceramide treatment induced autophagy in human trophoblast cells via a shift of the BOK-MCL1 rheostat toward prodeath BOK. Pharmacological inhibition of ASAH1 activity in pregnant mice resulted in increased placental ceramide content, abnormal placentation, reduced fetal growth, and increased autophagy via a similar shift in the BOK-MCL1 system. Our results reveal that oxidative stress-induced reduction of lysosomal hydrolase activities in combination with elevated de novo synthesis leads to ceramide overload, resulting in increased trophoblast cell autophagy, and typifies preeclampsia as a sphingolipid storage disorder.

  9. Cellulose Synthesis and Its Regulation

    PubMed Central

    Li, Shundai; Bashline, Logan; Lei, Lei; Gu, Ying

    2014-01-01

    Cellulose, the most abundant biopolymer synthesized on land, is made of linear chains of ß (1–4) linked D-glucose. As a major structural component of the cell wall, cellulose is important not only for industrial use but also for plant growth and development. Cellulose microfibrils are tethered by other cell wall polysaccharides such as hemicellulose, pectin, and lignin. In higher plants, cellulose is synthesized by plasma membrane-localized rosette cellulose synthase complexes. Despite the recent advances using a combination of molecular genetics, live cell imaging, and spectroscopic tools, many aspects of the cellulose synthesis remain a mystery. In this chapter, we highlight recent research progress towards understanding the mechanism of cellulose synthesis in Arabidopsis. PMID:24465174

  10. Loss of Frataxin activates the iron/sphingolipid/PDK1/Mef2 pathway in mammals.

    PubMed

    Chen, Kuchuan; Ho, Tammy Szu-Yu; Lin, Guang; Tan, Kai Li; Rasband, Matthew N; Bellen, Hugo J

    2016-11-30

    Friedreich's ataxia (FRDA) is an autosomal recessive neurodegenerative disease caused by mutations in Frataxin (FXN). Loss of FXN causes impaired mitochondrial function and iron homeostasis. An elevated production of reactive oxygen species (ROS) was previously proposed to contribute to the pathogenesis of FRDA. We recently showed that loss of frataxin homolog (fh), a Drosophila homolog of FXN, causes a ROS independent neurodegeneration in flies (Chen et al., 2016). In fh mutants, iron accumulation in the nervous system enhances the synthesis of sphingolipids, which in turn activates 3-phosphoinositide dependent protein kinase-1 (Pdk1) and myocyte enhancer factor-2 (Mef2) to trigger neurodegeneration of adult photoreceptors. Here, we show that loss of Fxn in the nervous system in mice also activates an iron/sphingolipid/PDK1/Mef2 pathway, indicating that the mechanism is evolutionarily conserved. Furthermore, sphingolipid levels and PDK1 activity are also increased in hearts of FRDA patients, suggesting that a similar pathway is affected in FRDA.

  11. Establishment of HeLa Cell Mutants Deficient in Sphingolipid-Related Genes Using TALENs

    PubMed Central

    Yamaji, Toshiyuki; Hanada, Kentaro

    2014-01-01

    Sphingolipids are essential components in eukaryotes and have various cellular functions. Recent developments in genome-editing technologies have facilitated gene disruption in various organisms and cell lines. We here show the disruption of various sphingolipid metabolic genes in human cervical carcinoma HeLa cells by using transcription activator-like effector nucleases (TALENs). A TALEN pair targeting the human CERT gene (alternative name COL4A3BP) encoding a ceramide transport protein induced a loss-of-function phenotype in more than 60% of HeLa cells even though the cell line has a pseudo-triploid karyotype. We have isolated several loss-of-function mutant clones for CERT, UGCG (encoding glucosylceramide synthase), and B4GalT5 (encoding the major lactosylceramide synthase), and also a CERT/UGCG double-deficient clone. Characterization of these clones supported previous proposals that CERT primarily contributes to the synthesis of SM but not GlcCer, and that B4GalT5 is the major LacCer synthase. These newly established sphingolipid-deficient HeLa cell mutants together with our previously established stable transfectants provide a ‘sphingolipid-modified HeLa cell panel,’ which will be useful to elucidate the functions of various sphingolipid species against essentially the same genomic background. PMID:24498430

  12. Protein Synthesis Initiation Factors: Phosphorylation and Regulation

    SciTech Connect

    Karen S. Browning

    2009-06-15

    The initiation of the synthesis of proteins is a fundamental process shared by all living organisms. Each organism has both shared and unique mechanisms for regulation of this vital process. Higher plants provide for a major amount of fixation of carbon from the environment and turn this carbon into food and fuel sources for our use. However, we have very little understanding of how plants regulate the synthesis of the proteins necessary for these metabolic processes. The research carried out during the grant period sought to address some of these unknowns in the regulation of protein synthesis initiation. Our first goal was to determine if phosphorylation plays a significant role in plant initiation of protein synthesis. The role of phosphorylation, although well documented in mammalian protein synthesis regulation, is not well studied in plants. We showed that several of the factors necessary for the initiation of protein synthesis were targets of plant casein kinase and showed differential phosphorylation by the plant specific isoforms of this kinase. In addition, we identified and confirmed the phosphorylation sites in five of the plant initiation factors. Further, we showed that phosphorylation of one of these factors, eIF5, affected the ability of the factor to participate in the initiation process. Our second goal was to develop a method to make initiation factor 3 (eIF3) using recombinant methods. To date, we successfully cloned and expressed 13/13 subunits of wheat eIF3 in E. coli using de novo gene construction methods. The final step in this process is to place the subunits into three different plasmid operons for co-expression. Successful completion of expression of eIF3 will be an invaluable tool to the plant translation community.

  13. Mechanism and regulation of eukaryotic protein synthesis.

    PubMed Central

    Merrick, W C

    1992-01-01

    This review presents a description of the numerous eukaryotic protein synthesis factors and their apparent sequential utilization in the processes of initiation, elongation, and termination. Additionally, the rare use of reinitiation and internal initiation is discussed, although little is known biochemically about these processes. Subsequently, control of translation is addressed in two different settings. The first is the global control of translation, which is effected by protein phosphorylation. The second is a series of specific mRNAs for which there is a direct and unique regulation of the synthesis of the gene product under study. Other examples of translational control are cited but not discussed, because the general mechanism for the regulation is unknown. Finally, as is often seen in an active area of investigation, there are several observations that cannot be readily accommodated by the general model presented in the first part of the review. Alternate explanations and various lines of experimentation are proposed to resolve these apparent contradictions. PMID:1620067

  14. Drug target identification in sphingolipid metabolism by computational systems biology tools: metabolic control analysis and metabolic pathway analysis.

    PubMed

    Ozbayraktar, F Betül Kavun; Ulgen, Kutlu O

    2010-08-01

    Sphingolipids regulate cellular processes that are critically important in cell's fate and function in cancer development and progression. This fact underlies the basics of the novel cancer therapy approach. The pharmacological manipulation of the sphingolipid metabolism in cancer therapeutics necessitates the detailed understanding of the pathway. Two computational systems biology tools are used to identify potential drug target enzymes among sphingolipid pathway that can be further utilized in drug design studies for cancer therapy. The enzymes in sphingolipid pathway were ranked according to their roles in controlling the metabolic network by metabolic control analysis. The physiologically connected reactions, i.e. biologically significant and functional modules of network, were identified by metabolic pathway analysis. The final set of candidate drug target enzymes are selected such that their manipulation leads to ceramide accumulation and long chain base phosphates depletion. The mathematical tools' efficiency for drug target identification performed in this study is validated by clinically available drugs.

  15. Tools for the analysis of metabolic flux through the sphingolipid pathway.

    PubMed

    Martínez-Montañés, Fernando; Schneiter, Roger

    2016-11-01

    Discerning the complex regulation of the enzymatic steps necessary for sphingolipid biosynthesis is facilitated by the utilization of tracers that allow a time-resolved analysis of the pathway dynamics without affecting the metabolic flux. Different strategies have been used and new tools are continuously being developed to probe the various enzymatic conversions that occur within this complex pathway. Here, we provide a short overview of the divergent fungal and mammalian sphingolipid biosynthetic routes, and of the tracers and methods that are frequently employed to follow the flux of intermediates throughout these pathways.

  16. Alteration of complex sphingolipid composition and its physiological significance in yeast Saccharomyces cerevisiae lacking vacuolar ATPase.

    PubMed

    Tani, Motohiro; Toume, Moeko

    2015-12-01

    In the yeast Saccharomyces cerevisiae, complex sphingolipids have three types of polar head group and five types of ceramide; however, the physiological significance of the structural diversity is not fully understood. Here, we report that deletion of vacuolar H+-ATPase (V-ATPase) in yeast causes dramatic alteration of the complex sphingolipid composition, which includes decreases in hydroxylation at the C-4 position of long-chain bases and the C-2 position of fatty acids in the ceramide moiety, decreases in inositol phosphorylceramide (IPC) levels, and increases in mannosylinositol phosphorylceramide (MIPC) and mannosyldiinositol phosphorylceramide [M(IP)2C] levels. V-ATPase-deleted cells exhibited slow growth at pH 7.2, whereas the increase in MIPC levels was significantly enhanced when V-ATPase-deleted cells were incubated at pH 7.2. The protein expression levels of MIPC and M(IP)2C synthases were significantly increased in V-ATPase-deleted cells incubated at pH 7.2. Loss of MIPC synthesis or an increase in the hydroxylation level of the ceramide moiety of sphingolipids on overexpression of Scs7 and Sur2 sphingolipid hydroxylases enhanced the growth defect of V-ATPase-deleted cells at pH 7.2. On the contrary, the growth rate of V-ATPase-deleted cells was moderately increased on the deletion of SCS7 and SUR2. In addition, supersensitivities to Ca2+, Zn2+ and H2O2, which are typical phenotypes of V-ATPase-deleted cells, were enhanced by the loss of MIPC synthesis. These results indicate the possibility that alteration of the complex sphingolipid composition is an adaptation mechanism for a defect of V-ATPase.

  17. Sphingolipids Mediate Differential Echinocandin Susceptibility in Candida albicans and Aspergillus nidulans

    PubMed Central

    Healey, Kelley R.; Challa, Krishna K.; Edlind, Thomas D.

    2015-01-01

    The cell wall synthesis-inhibiting echinocandins, including caspofungin and micafungin, play important roles in the treatment of candidiasis and aspergillosis. Previous studies revealed that, in the haploid yeast Candida glabrata, sphingolipid biosynthesis pathway mutations confer caspofungin reduced susceptibility (CRS) but micafungin increased susceptibility (MIS). Here, we describe one Candida albicans strain (of 10 tested) that similarly yields CRS-MIS mutants at relatively high frequency. Mutants demonstrated increased levels of long-chain bases (sphingolipid pathway intermediates) and, unique to this strain, loss of His104/Pro104 heterozygosity in the TSC13-encoded enoyl reductase. CRS-MIS was similarly observed in a C. albicans homozygous fen1Δ fen12Δ laboratory strain and in diverse wild-type strains following exogenous long-chain-base treatment. Analogous to these results, CRS-MIS was demonstrated in an Aspergillus nidulans basA mutant encoding defective sphingolipid C4-hydroxylase and in its wild-type parent exposed to long-chain bases. Sphingolipids likely modulate echinocandin interaction with their Fks membrane target in all susceptible fungi, with potential implications for optimizing therapy with existing antifungals and the development of novel agents. PMID:25824222

  18. Sphingolipids: A Potential Molecular Approach to Treat Allergic Inflammation

    PubMed Central

    Sun, Wai Y.; Bonder, Claudine S.

    2012-01-01

    Allergic inflammation is an immune response to foreign antigens, which begins within minutes of exposure to the allergen followed by a late phase leading to chronic inflammation. Prolonged allergic inflammation manifests in diseases such as urticaria and rhino-conjunctivitis, as well as chronic asthma and life-threatening anaphylaxis. The prevalence of allergic diseases is profound with 25% of the worldwide population affected and a rising trend across all ages, gender, and racial groups. The identification and avoidance of allergens can manage this disease, but this is not always possible with triggers being common foods, prevalent air-borne particles and only extremely low levels of allergen exposure required for sensitization. Patients who are sensitive to multiple allergens require prophylactic and symptomatic treatments. Current treatments are often suboptimal and associated with adverse effects, such as the interruption of cognition, sleep cycles, and endocrine homeostasis, all of which affect quality of life and are a financial burden to society. Clearly, a better therapeutic approach for allergic diseases is required. Herein, we review the current knowledge of allergic inflammation and discuss the role of sphingolipids as potential targets to regulate inflammatory development in vivo and in humans. We also discuss the benefits and risks of using sphingolipid inhibitors. PMID:23316248

  19. Plant sphingolipids: decoding the enigma of the Sphinx

    PubMed Central

    Pata, Mickael O.; Hannun, Yusuf A.; Ng, Carl K.-Y.

    2009-01-01

    Summary Sphingolipids are a ubiquitous class of lipids present in a variety of organisms including eukaryotes and bacteria. In the last two decades, research has focused on characterizing the individual species of this complex family of lipids, leading to a new field of research called sphingolipidomics. There are at least 500 (and perhaps thousands) different molecular species of sphingolipids in cells, and in Arabidopsis alone, it has been reported that there are at least 168 different sphingolipids. Plant sphingolipids can be divided into four classes: glycosyl inositol phosphoceramides (GIPCs), glycosylceramides, ceramides, and free long chain bases (LCBs). Numerous enzymes involved in plant sphingolipid metabolism have now been cloned and characterized, and, in general, there is broad conservation in the way sphingolipids are metabolized in animals, yeast and plants. Here, we review the diversity of sphingolipids reported in the literature, some of the recent advances in our understanding of sphingolipid metabolism in plants, and the physiological roles that sphingolipids and sphingolipid metabolites play in plant physiology. PMID:20028469

  20. A sphingolipid mechanism for behavioral extinction.

    PubMed

    Huston, Joseph P; Kornhuber, Johannes; Mühle, Christiane; Japtok, Lukasz; Komorowski, Mara; Mattern, Claudia; Reichel, Martin; Gulbins, Erich; Kleuser, Burkhard; Topic, Bianca; De Souza Silva, Maria A; Müller, Christian P

    2016-05-01

    Reward-dependent instrumental behavior must continuously be re-adjusted according to environmental conditions. Failure to adapt to changes in reward contingencies may incur psychiatric disorders like anxiety and depression. When an expected reward is omitted, behavior undergoes extinction. While extinction involves active re-learning, it is also accompanied by emotional behaviors indicative of frustration, anxiety, and despair (extinction-induced depression). Here, we report evidence for a sphingolipid mechanism in the extinction of behavior. Rapid extinction, indicating efficient re-learning, coincided with a decrease in the activity of the enzyme acid sphingomyelinase (ASM), which catalyzes turnover of sphingomyelin to ceramide, in the dorsal hippocampus of rats. The stronger the decline in ASM activity, the more rapid was the extinction. Sphingolipid-focused lipidomic analysis showed that this results in a decline of local ceramide species in the dorsal hippocampus. Ceramides shape the fluidity of lipid rafts in synaptic membranes and by that way can control neural plasticity. We also found that aging modifies activity of enzymes and ceramide levels in selective brain regions. Aging also changed how the chronic treatment with corticosterone (stress) or intranasal dopamine modified regional enzyme activity and ceramide levels, coinciding with rate of extinction. These data provide first evidence for a functional ASM-ceramide pathway in the brain involved in the extinction of learned behavior. This finding extends the known cellular mechanisms underlying behavioral plasticity to a new class of membrane-located molecules, the sphingolipids, and their regulatory enzymes, and may offer new treatment targets for extinction- and learning-related psychopathological conditions. Sphingolipids are common lipids in the brain which form lipid domains at pre- and postsynaptic membrane compartments. Here we show a decline in dorsal hippocampus ceramide species together with a

  1. Phase behavior of carbamyloxyphosphatidylcholine, a sphingolipid analogue.

    PubMed

    Curatolo, W; Bali, A; Gupta, C M

    1985-12-01

    The phase behavior of two series of phosphatidylcholines (PC) possessing carbamyloxy-linked fatty acids has been studied by differential scanning calorimetry. These non-natural phosphatidylcholines are of interest because they possess the capability of forming interlipid hydrogen bonds via the carbamyloxy function and as such can serve as a model for sphingolipids, the phase behavior of which is thought to be dominated by interlipid hydrogen bonding. Furthermore, carbamyloxyphosphatidylcholines form unusually stable liposomes, and thus have potential in drug delivery. Carbamyloxyphosphatidylcholines of the type di-(CnH2n+1NHCOO)-PC, where n = 13, 15, or 17, exhibit metastable polymorphism. Cooling from the liquid crystalline state results in formation of a metastable low-temperature polymorph I, which must transform into a stable low-temperature polymorph II before the liquid crystalline state can be reached again. Carbamyloxyphosphatidylcholines of the type 1-C16:0-2-(CnH2n+1NHCOO)-PC exhibit similar metastable polymorphism. This metastability is similar to that exhibited by certain cerebrosides and sphingomyelins and indicates that the sphingosine backbone is not a prerequisite for this type of metastability. Furthermore, the carbamyloxy group is reversed in orientation compared with the amide of sphingolipids (-NHCO- versus -CONH-), suggesting that the intermolecular hydrogen bonding potential, rather than some highly specific steric or conformational constraint, is responsible for the observed metastability of sphingolipids.

  2. Sphingolipid metabolism is strikingly different between pollen and leaf in Arabidopsis as revealed by compositional and gene expression profiling.

    PubMed

    Luttgeharm, Kyle D; Kimberlin, Athen N; Cahoon, Rebecca E; Cerny, Ronald L; Napier, Johnathan A; Markham, Jonathan E; Cahoon, Edgar B

    2015-07-01

    Although sphingolipids are essential for male gametophytic development in Arabidopsis thaliana, sphingolipid composition and biosynthetic gene expression have not been previously examined in pollen. In this report, electrospray ionization (ESI)-MS/MS was applied to characterization of sphingolipid compositional profiles in pollen isolated from wild type Arabidopsis Col-0 and a long-chain base (LCB) Δ4 desaturase mutant. Pollen fractions were highly enriched in glucosylceramides (GlcCer) relative to levels previously reported in leaves. Accompanying the loss of the Δ4 unsaturated LCB sphingadiene (d18:2) in the Δ4 desaturase mutant was a 50% reduction in GlcCer concentrations. In addition, pollen glycosylinositolphosphoceramides (GIPCs) were found to have a complex array of N-acetyl-glycosylated GIPCs, including species with up to three pentose units that were absent from leaf GIPCs. Underlying the distinct sphingolipid composition of pollen, genes for key biosynthetic enzymes for GlcCer and d18:2 synthesis and metabolism were more highly expressed in pollen than in leaves or seedlings, including genes for GlcCer synthase (GCS), sphingoid base C-4 hydroxylase 2 (SBH2), LCB Δ8 desaturases (SLD1 and SLD2), and LOH2 ceramide synthase (LOH2). Overall, these findings indicate strikingly divergent sphingolipid metabolism between pollen and leaves in Arabidopsis, the significance of which remains to be determined.

  3. Yeast lacking the amphiphysin family protein Rvs167 is sensitive to disruptions in sphingolipid levels.

    PubMed

    Toume, Moeko; Tani, Motohiro

    2016-08-01

    Rvs167 and Rvs161 in Saccharomyces cerevisiae are amphiphysin family proteins, which are involved in several important cellular events, such as invagination and scission of endocytic vesicles, and actin cytoskeleton organization. It has been reported that cellular dysfunctions caused by deletion of RVS167 or RVS161 are rescued by deletion of specific nonessential sphingolipid-metabolizing enzyme genes. Here, we found that yeast cells lacking RVS167 or RVS161 exhibit a decrease in sphingolipid levels. In rvs167∆ cells, the expression level of Orm2, a negative regulator of serine palmitoyltransferase (SPT) catalyzing the initial step of sphingolipid biosynthesis, was increased in a calcineurin-dependent manner, and the decrease in sphingolipid levels in rvs167∆ cells was reversed on deletion of ORM2. Moreover, repression of both ORM1 and ORM2 expression or overexpression of SPT caused a strong growth defect of rvs167∆ cells, indicating that enhancement of de novo sphingolipid biosynthesis is detrimental to rvs167∆ cells. In contrast, partial repression of LCB1-encoding SPT suppressed abnormal phenotypes caused by the deletion of RVS167, including supersensitivity to high temperature and salt stress, and impairment of endocytosis and actin cytoskeleton organization. In addition, the partial repression of SPT activity suppressed the temperature supersensitivity and abnormal vacuolar morphology caused by deletion of VPS1 encoding a dynamin-like GTPase, which is required for vesicle scission and is functionally closely related to Rvs167/Rvs161, whereas repression of both ORM1 and ORM2 expression in vps1∆ cells caused a growth defect. Thus, it was suggested that proper regulation of SPT activity is indispensable for amphiphysin-deficient cells.

  4. Orosomucoid Proteins Interact with the Small Subunit of Serine Palmitoyltransferase and Contribute to Sphingolipid Homeostasis and Stress Responses in Arabidopsis[OPEN

    PubMed Central

    Li, Jian; Yin, Jian; Rong, Chan; Li, Kai-En; Wu, Jian-Xin; Huang, Li-Qun; Zeng, Hong-Yun; Sahu, Sunil Kumar; Yao, Nan

    2016-01-01

    Serine palmitoyltransferase (SPT), a pyridoxyl-5′-phosphate-dependent enzyme, catalyzes the first and rate-limiting step in sphingolipid biosynthesis. In humans and yeast, orosomucoid proteins (ORMs) negatively regulate SPT and thus play an important role in maintaining sphingolipid levels. Despite the importance of sphingoid intermediates as bioactive molecules, the regulation of sphingolipid biosynthesis through SPT is not well understood in plants. Here, we identified and characterized the Arabidopsis thaliana ORMs, ORM1 and ORM2. Loss of function of both ORM1 and ORM2 (orm1 amiR-ORM2) stimulated de novo sphingolipid biosynthesis, leading to strong sphingolipid accumulation, especially of long-chain bases and ceramides. Yeast two-hybrid, bimolecular fluorescence complementation, and coimmunoprecipitation assays confirmed that ORM1 and ORM2 physically interact with the small subunit of SPT (ssSPT), indicating that ORMs inhibit ssSPT function. We found that orm1 amiR-ORM2 plants exhibited an early-senescence phenotype accompanied by H2O2 production at the cell wall and in mitochondria, active vesicular trafficking, and formation of cell wall appositions. Strikingly, the orm1 amiR-ORM2 plants showed increased expression of genes related to endoplasmic reticulum stress and defenses and also had enhanced resistance to oxidative stress and pathogen infection. Taken together, our findings indicate that ORMs interact with SPT to regulate sphingolipid homeostasis and play a pivotal role in environmental stress tolerance in plants. PMID:27923879

  5. Specific sphingolipid content decrease in Cerkl knockdown mouse retinas

    PubMed Central

    Garanto, Alejandro; Mandal, Nawajes A.; Egido-Gabás, Meritxell; Marfany, Gemma; Fabriàs, Gemma; Anderson, Robert E.; Casas, Josefina; Gonzàlez-Duarte, Roser

    2014-01-01

    Sphingolipids (SPLs) are finely tuned structural compounds and bioactive molecules involved in membrane fluidity and cellular homeostasis. The core sphingolipid, ceramide (CER), and its derivatives, regulate several crucial processes in neuronal cells, among them cell differentiation, cell–cell interactions, membrane conductance, synaptic transmission, and apoptosis. Mutations in Ceramide Kinase-Like (CERKL) cause autosomal recessive Retinitis Pigmentosa and Cone Rod Dystrophy. The presence of a conserved lipid kinase domain and the overall similarity with CERK suggested that CERKL might play a role in the SPL metabolism as a CER kinase. Unfortunately, CERKL function and substrate(s), as well as its contribution to the retinal etiopathology, remain as yet unknown. In this work we aimed to characterize the mouse retinal sphingolipidome by UPLC-TOF to first, thoroughly investigate the SPL composition of the murine retina, compare it to our Cerkl −/− model, and finally assess new possible CERKL substrates by phosphorus quantification and protein-lipid overlay. Our results showed a consistent and notable decrease of the retinal SPL content (mainly ranging from 30% to 60%) in the Cerkl −/− compared to WT retinas, which was particularly evident in the glucosyl/galactosyl ceramide species (Glc/GalCer) whereas the phospholipids and neutral lipids remained unaltered. Moreover, evidence in favor of CERKL binding to GlcCer, GalCer and sphingomyelin has been gathered. Altogether, these results highlight the involvement of CERKL in the SPL metabolism, question its role as a kinase, and open new scenarios concerning its function. PMID:23501591

  6. Loss of Frataxin activates the iron/sphingolipid/PDK1/Mef2 pathway in mammals

    PubMed Central

    Chen, Kuchuan; Ho, Tammy Szu-Yu; Lin, Guang; Tan, Kai Li; Rasband, Matthew N; Bellen, Hugo J

    2016-01-01

    Friedreich’s ataxia (FRDA) is an autosomal recessive neurodegenerative disease caused by mutations in Frataxin (FXN). Loss of FXN causes impaired mitochondrial function and iron homeostasis. An elevated production of reactive oxygen species (ROS) was previously proposed to contribute to the pathogenesis of FRDA. We recently showed that loss of frataxin homolog (fh), a Drosophila homolog of FXN, causes a ROS independent neurodegeneration in flies (Chen et al., 2016). In fh mutants, iron accumulation in the nervous system enhances the synthesis of sphingolipids, which in turn activates 3-phosphoinositide dependent protein kinase-1 (Pdk1) and myocyte enhancer factor-2 (Mef2) to trigger neurodegeneration of adult photoreceptors. Here, we show that loss of Fxn in the nervous system in mice also activates an iron/sphingolipid/PDK1/Mef2 pathway, indicating that the mechanism is evolutionarily conserved. Furthermore, sphingolipid levels and PDK1 activity are also increased in hearts of FRDA patients, suggesting that a similar pathway is affected in FRDA. DOI: http://dx.doi.org/10.7554/eLife.20732.001 PMID:27901468

  7. Ubiquinone synthesis and its regulation in Pneumocystis carinii.

    PubMed

    Kaneshiro, Edna S; Basselin, Mireille; Merali, Salim; Kayser, Oliver

    2006-01-01

    The opportunistic pathogen Pneumocystis causes a type of pneumonia in individuals with defective immune systems such as AIDS patients. Atovaquone, an analog of ubiquinone (coenzyme Q [CoQ]), is effective in clearing mild to moderate cases of the infection. Rat-derived Pneumocystis carinii was the first organism in which CoQ synthesis was clearly demonstrated to occur in both mitochondrial and microsomal subcellular fractions. Atovaquone inhibits microsomal CoQ synthesis with no effect on mitochondrial CoQ synthesis. We here report on additional studies evaluating CoQ synthesis and its regulation in the organism. Buparvaquone also inhibited CoQ synthesis and it reduced the synthesis of all four CoQ homologs in the microsomal but not the mitochondrial fraction. Glyphosate, which inhibits a reaction in the de novo synthesis of the benzoquinone moiety of CoQ reduced cellular ATP levels. Bacterial and plant quinones, and several chemically synthesized phenolics, flavanoids, and naphthoquinones that inhibit electron transport in other organisms were shown to reduce CoQ synthesis in P. carinii. The inhibitory action of naphthoquinone compounds appeared to depend on their molecular size and structural flexibility rather than redox potential. Results of experiments examining the synthesis of the polyprenyl chain of CoQ were consistent with negative feedback control of CoQ synthesis. These studies on P. carinii suggest that cellular sites and the control of CoQ synthesis in different organisms and cell types might be more diverse than previously thought.

  8. Sphingolipids as Mediators in the Crosstalk between Microbiota and Intestinal Cells: Implications for Inflammatory Bowel Disease.

    PubMed

    Bryan, Phillips-Farfán; Karla, Carvajal; Edgar Alejandro, Medina-Torres; Sara Elva, Espinosa-Padilla; Gemma, Fabrias; Luz, Camacho

    Inflammatory bowel disease (IBD) describes different illnesses characterized by chronic inflammation of the gastrointestinal tract. Although the pathogenic mechanisms leading to IBD are poorly understood, immune system disturbances likely underlie its development. Sphingolipids (SLs) have been identified as important players and promising therapeutic targets to control inflammation in IBD. Interestingly, it seems that microorganisms of the normal gut microbiota and probiotics are involved in sphingolipid function. However, there is a great need to investigate the role of SLs as intermediates in the crosstalk between intestinal immunity and microorganisms. This review focuses on recent investigations that describe some mechanisms involved in the regulation of cytokine profiles by SLs. We also describe the importance of gut microbiota in providing signaling molecules that favor the communication between resident bacteria and intestinal cells. This, in turn, modulates the immune response in the bowel and likely in other peripheral organs. The potential of SLs and gut microbiota as targets or therapeutic agents for IBD is also discussed.

  9. Regulation of Phospholipid Synthesis in the Yeast Saccharomyces cerevisiae

    PubMed Central

    Carman, George M.; Han, Gil-Soo

    2013-01-01

    The yeast Saccharomyces cerevisiae, with its full complement of organelles, synthesizes membrane phospholipids by pathways that are generally common to those found in higher eukaryotes. Phospholipid synthesis in yeast is regulated in response to a variety of growth conditions (e.g., inositol supplementation, zinc depletion, and growth stage) by a coordination of genetic (e.g., transcriptional activation and repression) and biochemical (e.g., activity modulation and localization) mechanisms. Phosphatidate (PA), whose cellular levels are controlled by the activities of key phospholipid synthesis enzymes, plays a central role in the transcriptional regulation of phospholipid synthesis genes. In addition to the regulation of gene expression, phosphorylation of key phospholipid synthesis catalytic and regulatory proteins controls the metabolism of phospholipid precursors and products. PMID:21275641

  10. Analysis of Mammalian Sphingolipids by Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) and Tissue Imaging Mass Spectrometry (TIMS)

    PubMed Central

    Sullards, M. Cameron; Liu, Ying; Chen, Yanfeng; Merrill, Alfred H.

    2011-01-01

    Sphingolipids are a highly diverse category of molecules that serve not only as components of biological structures but also as regulators of numerous cell functions. Because so many of the structural features of sphingolipids give rise to their biological activity, there is a need for comprehensive or “sphingolipidomic” methods for identification and quantitation of as many individual subspecies as possible. This review defines sphingolipids as a class, briefly discusses classical methods for their analysis, and focuses primarily on liquid chromatography tandem mass spectrometry (LC-MS/MS) and tissue imaging mass spectrometry (TIMS). Recently, a set of evolving and expanding methods have been developed and rigorously validated for the extraction, identification, separation, and quantitation of sphingolipids by LC-MS/MS. Quantitation of these biomolecules is made possible via the use of an internal standard cocktail. The compounds that can be readily analyzed are free long-chain (sphingoid) bases, sphingoid base 1-phosphates, and more complex species such as ceramides, ceramide 1-phosphates, sphingomyelins, mono- and di-hexosylceramides sulfatides, and novel compounds such as the 1-deoxy- and 1-(deoxymethyl)-sphingoid bases and their N-acyl-derivatives. These methods can be altered slightly to separate and quantitate isomeric species such as glucosyl/galactosylceramide. Because these techniques require the extraction of sphingolipids from their native environment, any information regarding their localization in histological slices is lost. Therefore, this review also describes methods for TIMS. This technique has been shown to be a powerful tool to determine the localization of individual molecular species of sphingolipids directly from tissue slices. PMID:21749933

  11. Sphingolipids in Genetic and Acquired Forms of Chronic Kidney Diseases.

    PubMed

    Ueda, Norishi

    2017-01-12

    Sphingolipids (SLs) regulate apoptosis, proliferation, and stress response. SLs, including ceramide, glycosphingolipids (glucosylceramide, lactosylceramide, and gangliosides) and sphingosine-1-phosphate (S1P), play a role in the pathogenesis and progression of genetic (lysosomal storage disease, congenital nephrotic syndrome and polycystic kidney disease) and non-genetic forms of chronic kidney diseases (CKDs). SLs metabolism defects promote complications (cardiovascular events, etc.) via oxidant stress in CKDs. A balancing role of apoptotic SLs and anti-apoptotic S1P is crucial in the regulation of glomerular injury and complications associated with CKDs. Interaction between SLs, endothelial function and renin-angiotensin-aldosterone system (RAAS) plays an important role in the regulation of glomerular injury. SLs affect mitochondrial function that regulate the opening of mitochondrial permeability transition (MPT) pore, mitochondrial outer membrane permeability (MOMP), generation of reactive oxygen species (ROS), Bcl-2 family proteins, leading to cytochrome c release and caspase activation, leading to apoptosis, and regulate glomerular cell proliferation or renal fibrosis. Interaction between SLs, endothelial function and RAAS plays a role in the regulation of glomerular injury. This review article summarizes the current evidence supporting a role of SLs metabolism defects in the pathogenesis and progression of glomerular injury and discusses a role of mitochondria, including MPT pore, MOMP, ROS, Bcl-2 family proteins, interaction between SLs, endothelial function and RAAS, and SLs-induced downstream signaling events in CKDs. Crosstalk between these factors plays a role in the pathogenesis and progression of CKDs. Therapeutic strategy of targeting SLs metabolism defects for CKDs through modulation of the enzymes responsible for SLs metabolism defects are also discussed.

  12. Iron, Glucose and Intrinsic Factors Alter Sphingolipid Composition as Yeast Cells Enter Stationary Phase

    PubMed Central

    Lester, Robert L.; Withers, Bradley R.; Schultz, Megan A.; Dickson, Robert C.

    2013-01-01

    Survival of Saccharomyces cerevisiae cells, like most microorganisms, requires switching from a rapidly dividing to a non-dividing or stationary state. To further understand how cells navigate this switch, we examined sphingolipids since they are key structural elements of membranes and also regulate signaling pathways vital for survival. During and after the switch to a non-dividing state there is a large increase in total free and sphingolipid-bound long chain-bases and an even larger increase in free and bound C20-long-chain bases, which are nearly undetectable in dividing cells. These changes are due to intrinsic factors including Orm1 and Orm2, ceramide synthase, Lcb4 kinase and the Tsc3 subunit of serine palmitoyltransferase as well as extrinsic factors including glucose and iron. Lowering the concentration of glucose, a form of calorie restriction, decreases the level of LCBs, which is consistent with the idea that reducing the level of some sphingolipids enhances lifespan. In contrast, iron deprivation increases LCB levels and decreases long term survival; however, these phenomena may not be related because iron deprivation disrupts many metabolic pathways. The correlation between increased LCBs and shorter lifespan is unsupported at this time. The physiological rise in LCBs that we observe may serve to modulate nutrient transporters and possibly other membrane phenomena that contribute to enhanced stress resistance and survival in stationary phase. PMID:23286903

  13. Posttranscriptional regulation of pineal melatonin synthesis in Octodon degus.

    PubMed

    Lee, Soo Jung; Liu, Tiecheng; Chattoraj, Asamanja; Zhang, Samantha L; Wang, Lijun; Lee, Theresa M; Wang, Michael M; Borjigin, Jimo

    2009-08-01

    Small laboratory animals have provided significant information about melatonin regulation, yet most of these organisms are nocturnal and regulate melatonin synthesis by mechanisms that diverge from those of humans. For example, in all rodents examined, melatonin secretion occurs with a time lag of several hours after the onset of darkness; in addition, arylalkylamine N-acetyltransferase (AANAT), the key enzyme in melatonin synthesis, displays dynamic transcriptional activation specifically at night in all rodents studied to date. In ungulates and primates including humans, on the other hand, melatonin secretion occurs immediately during the early night and is controlled by circadian posttranscriptional regulation of AANAT. We hypothesize that the diurnal Octodon degus (an Hystricognath rodent) could serve as an improved experimental model for studies of human melatonin regulation. To test this, we monitored melatonin production in degus using pineal microdialysis and characterized the regulation of melatonin synthesis by analyzing degu Aanat. Degu pineal melatonin rises with little latency at night, as in ungulates and primates. In addition, degu Aanat mRNA expression displays no detectable diurnal variation, suggesting that, like ungulates and primates, melatonin in this species is regulated by a posttranscriptional mechanism. Compared with AANAT from all rodents examined to date, the predicted amino acid sequence of degu AANAT is phylogenetically more closely related to ungulate and primate AANAT. These data suggest that Octodon degus may provide an ideal model system for laboratory investigation of mechanisms of melatonin synthesis and secretion in diurnal mammals.

  14. Sphingolipid biosynthesis upregulation by TOR complex 2-Ypk1 signaling during yeast adaptive response to acetic acid stress.

    PubMed

    Guerreiro, Joana F; Muir, Alexander; Ramachandran, Subramaniam; Thorner, Jeremy; Sá-Correia, Isabel

    2016-12-01

    Acetic acid-induced inhibition of yeast growth and metabolism limits the productivity of industrial fermentation processes, especially when lignocellulosic hydrolysates are used as feedstock in industrial biotechnology. Tolerance to acetic acid of food spoilage yeasts is also a problem in the preservation of acidic foods and beverages. Thus understanding the molecular mechanisms underlying adaptation and tolerance to acetic acid stress is increasingly important in industrial biotechnology and the food industry. Prior genetic screens for Saccharomyces cerevisiae mutants with increased sensitivity to acetic acid identified loss-of-function mutations in the YPK1 gene, which encodes a protein kinase activated by the target of rapamycin (TOR) complex 2 (TORC2). We show in the present study by several independent criteria that TORC2-Ypk1 signaling is stimulated in response to acetic acid stress. Moreover, we demonstrate that TORC2-mediated Ypk1 phosphorylation and activation is necessary for acetic acid tolerance, and occurs independently of Hrk1, a protein kinase previously implicated in the cellular response to acetic acid. In addition, we show that TORC2-Ypk1-mediated activation of l-serine:palmitoyl-CoA acyltransferase, the enzyme complex that catalyzes the first committed step of sphingolipid biosynthesis, is required for acetic acid tolerance. Furthermore, analysis of the sphingolipid pathway using inhibitors and mutants indicates that it is production of certain complex sphingolipids that contributes to conferring acetic acid tolerance. Consistent with that conclusion, promoting sphingolipid synthesis by adding exogenous long-chain base precursor phytosphingosine to the growth medium enhanced acetic acid tolerance. Thus appropriate modulation of the TORC2-Ypk1-sphingolipid axis in industrial yeast strains may have utility in improving fermentations of acetic acid-containing feedstocks.

  15. Regulation of cholesterol synthesis in four colonic adenocarcinoma cell lines.

    PubMed

    Cerda, S R; Wilkinson, J; Broitman, S A

    1995-12-01

    Colon tumor cells, unlike normal human fibroblasts, exhibited an uncoupling of low density lipoprotein (LDL)-derived cholesterol from cellular growth, when endogenous cholesterol synthesis was inhibited by mevinolin, a hydroxymethylglutaryl-CoA reductase (HMG-CoAR) competitive inhibitor [Fabricant, M., and Broitman, S.A. (1990) Cancer Res. 50, 632-636]. Further evaluation of cholesterol metabolism was conducted in two undifferentiated (SW480, SW1417) and two differentiated (HT29, CACO2) colonic adenocarcinoma (adeno-CA) cell lines and an untransformed human fibroblast, AG1519A. Cells grown in monolayer culture to near subconfluency were used to assess endogenous cholesterol synthesis by 14C-acetate incorporation, in response to the following treatments in lipoprotein-deficient serum (LPDS)-supplemented minimum essential medium (MEM): LPDS alone, LDL, mevinolin, mevinolin with LDL, and 25-hydroxy-cholesterol (25-OH-CH). Complete fetal bovine serum (FBS)-supplemented MEM was used as control. All colon tumor lines exhibited similarly high endogenous cholesterol synthesis in both FBS and LPDS relative to the fibroblasts which demonstrated low basal levels in FBS and maximal synthesis in LPDS. LDL treatment did not inhibit cholesterol synthesis in colon tumor cells, but suppressed that in the fibroblast by 70%. Sterol repression of cholesterol synthesis mediated by 25-OH-CH occurred in all cells. Mevinolin caused a reduction in cholesterol synthesis in the colonic cancer cell lines, which was not further decreased by concurrent addition of LDL. In contrast, in mevinolin-treated fibroblasts, LDL further inhibited cholesterol synthesis. When the effect of cell density on cholesterol synthesis regulation was evaluated under conditions of sparse density in SW480 and SW147, results indicated that (i) basal rates of cholesterol synthesis were higher, (ii) LDL inhibited cholesterol synthesis more effectively, and (iii) mevinolin or 25-OH-CH had a more pronounced effect than in

  16. Lipoxygenase inhibitory sphingolipids from Launaea nudicaulis.

    PubMed

    Riaz, Naheed; Parveen, Shehla; Saleem, Muhammad; Ali, Muhammad Shaiq; Malik, Abdul; Ashraf, Muhammad; Afzal, Iftikhar; Jabbar, Abdul

    2012-01-01

    Four new sphingolipids: nudicaulin A [(2S,3S,4R,14E)-2-{[octadecanoyl]amino}tetraeicos-14-ene-1,3,4-triol; 1], nudicaulin B [(2S,3S,4R,14E)-2-{[(2R)-2-hydroxyoctadecanoyl]amino}tetraeicos-14-ene-1,3,4-triol; 2], nudicaulin C [(2S,3S,4R,14E)-2-{[(2R)-2-hydroxyoctadecanoyl]amino}tetraeicos-14-ene-1,3,4-triol-1-O-β-D-glucopyranoside; 3], and nudicaulin D [(2S,3S,4R)-2-{[(2R,3S,12E)-2,3-dihydroxyeicos-12-enoyl]amino}octadecane-1,3,4-triol; 4] together with 1-hexatriacontanol, β-sitosterol, octadecyl 4-hydroxycinnamate, elaidic acid, cholesta-5,22-diene-3,7-diol, oleanolic acid, apigenin, and β-sitosterol 3-O-β-D-glucopyranoside were isolated from the methanolic extract of the whole plant of Launaea nudicaulis. Their structures were elucidated using ¹H and ¹³C NMR spectra and 2D NMR analyses (HMQC, HMBC, and COSY) in combination with mass spectrometry (EI-MS, HR-EI-MS, FAB-MS, and HR-FAB-MS) experiments and comparison with literature data of related compounds. Compounds 1-4 displayed moderate inhibitory potential against enzyme lipoxygenase in concentration-dependent manner with IC₅₀ value ranges 103-193 μM.

  17. Mechanism and Regulation of Protein Synthesis in Saccharomyces cerevisiae.

    PubMed

    Dever, Thomas E; Kinzy, Terri Goss; Pavitt, Graham D

    2016-05-01

    In this review, we provide an overview of protein synthesis in the yeast Saccharomyces cerevisiae The mechanism of protein synthesis is well conserved between yeast and other eukaryotes, and molecular genetic studies in budding yeast have provided critical insights into the fundamental process of translation as well as its regulation. The review focuses on the initiation and elongation phases of protein synthesis with descriptions of the roles of translation initiation and elongation factors that assist the ribosome in binding the messenger RNA (mRNA), selecting the start codon, and synthesizing the polypeptide. We also examine mechanisms of translational control highlighting the mRNA cap-binding proteins and the regulation of GCN4 and CPA1 mRNAs.

  18. The sphingolipid biosynthetic enzyme Sphingolipid delta8 desaturase is important for chilling resistance of tomato

    PubMed Central

    Zhou, Ying; Zeng, Lanting; Fu, Xiumin; Mei, Xin; Cheng, Sihua; Liao, Yinyin; Deng, Rufang; Xu, Xinlan; Jiang, Yueming; Duan, Xuewu; Baldermann, Susanne; Yang, Ziyin

    2016-01-01

    The physiological functions of sphingolipids in animals have been intensively studied, while less attention has been paid to their roles in plants. Here, we reveal the involvement of sphingolipid delta8 desaturase (SlSLD) in the chilling resistance of tomato (Solanum lycopersicum cv. Micro-Tom). We used the virus-induced gene silencing (VIGS) approach to knock-down SlSLD expression in tomato leaves, and then evaluated chilling resistance. Changes in leaf cell structure under a chilling treatment were observed by transmission electron microscopy. In control plants, SlSLD was highly expressed in the fruit and leaves in response to a chilling treatment. The degree of chilling damage was greater in SlSLD-silenced plants than in control plants, indicating that SlSLD knock-down significantly reduced the chilling resistance of tomato. Compared with control plants, SlSLD-silenced plants showed higher relative electrolytic leakage and malondialdehyde content, and lower superoxide dismutase and peroxidase activities after a chilling treatment. Chilling severely damaged the chloroplasts in SlSLD-silenced plants, resulting in the disruption of chloroplast membranes, swelling of thylakoids, and reduced granal stacking. Together, these results show that SlSLD is crucial for chilling resistance in tomato. PMID:27929095

  19. Sphingolipid long-chain-base auxotrophs of Saccharomyces cerevisiae: genetics, physiology, and a method for their selection.

    PubMed Central

    Pinto, W J; Srinivasan, B; Shepherd, S; Schmidt, A; Dickson, R C; Lester, R L

    1992-01-01

    A selection method for sphingolipid long-chain-base auxotrophs of Saccharomyces cerevisiae was devised after observing that strains that require a long-chain base for growth become denser when starved for this substance. Genetic analysis of over 60 such strains indicated only two complementation classes, lcb1 and lcb2. Mutant strains from each class grew equally well with 3-ketodihydrosphingosine, erythrodihydrosphingosine or threodihydrosphingosine, or phytosphingosine. Since these metabolites represent the first, second, and last components, respectively, of the long-chain-base biosynthetic pathway, it is likely that the LCB1 and LCB2 genes are involved in the first step of long-chain-base synthesis. The results of long-chain-base starvation in the Lcb- strains suggest that one or more sphingolipids have a vital role in S. cerevisiae. Immediate sequelae of long-chain-base starvation were loss of viability, exacerbated in the presence of alpha-cyclodextrin, and loss of phosphoinositol sphingolipid synthesis but not phosphatidylinositol synthesis. Loss of viability with long-chain-base starvation could be prevented by also blocking either protein or nucleic acid synthesis. Without a long-chain-base, cell division, dry mass accumulation, and protein synthesis continued at a diminished rate and were further inhibited by the detergent Tergitol. The cell density increase induced by long-chain-base starvation is thus explained as a differential loss of cell division and mass accumulation. Long-chain-base starvation in Lcb- S. cerevisiae and inositol starvation of Inos- S. cerevisiae share common features: an increase in cell density and a loss of cell viability overcome by blocking macromolecular synthesis. PMID:1556075

  20. Regulation of starch synthesis in potato tubers

    SciTech Connect

    Davies, H.; Oparka, K.; Viola, R.; Wright, K.; Ross, H. )

    1990-05-01

    Following tuber excision from the mother plant sucrose synthase activity fell from 3,120 to 960 nmol/g.f. wt./h within 7 days and starch synthesis ({sup 14}C sucrose incorporated into isolated discs) from 23 to 7 nmol/g.f. wt./h. While the maximum catalytic activity of sucrose synthase was more than sufficient to account for the observed rate of starch synthesis a maximum of 27% of sucrose incorporated by discs was converted into starch within 3 h. This compared with 80% conversion of {sup 14}C glucose incorporated. Tuber excision also reduced the rate of starch biosynthesis with glucose as a substrate (from 206 to 64 nmol/g.f. wt./h). The activities of UDPG-pyrophosphorylase, PPi-PFK, ATP-PFK, starch synthase and hexokinase (glucose or fructose substrates) were unaffected by tuber removal. ADPG pyrophosphorylase activity was reduced from 8,000 to 4,500 nmol/g.f. wt./h. Preliminary experiments indicate that the decline in sucrose synthease activity is prevented by maintaining sucrose flux into tubers through the cut stolon.

  1. Disruption of Sphingolipid Biosynthesis Blocks Phagocytosis of Candida albicans.

    PubMed

    Tafesse, Fikadu G; Rashidfarrokhi, Ali; Schmidt, Florian I; Freinkman, Elizaveta; Dougan, Stephanie; Dougan, Michael; Esteban, Alexandre; Maruyama, Takeshi; Strijbis, Karin; Ploegh, Hidde L

    2015-10-01

    The ability of phagocytes to clear pathogens is an essential attribute of the innate immune response. The role of signaling lipid molecules such as phosphoinositides is well established, but the role of membrane sphingolipids in phagocytosis is largely unknown. Using a genetic approach and small molecule inhibitors, we show that phagocytosis of Candida albicans requires an intact sphingolipid biosynthetic pathway. Blockade of serine-palmitoyltransferase (SPT) and ceramide synthase-enzymes involved in sphingolipid biosynthesis- by myriocin and fumonisin B1, respectively, impaired phagocytosis by phagocytes. We used CRISPR/Cas9-mediated genome editing to generate Sptlc2-deficient DC2.4 dendritic cells, which lack serine palmitoyl transferase activity. Sptlc2-/- DC2.4 cells exhibited a stark defect in phagocytosis, were unable to bind fungal particles and failed to form a normal phagocytic cup to engulf C. albicans. Supplementing the growth media with GM1, the major ganglioside present at the cell surface, restored phagocytic activity of Sptlc2-/- DC2.4 cells. While overall membrane trafficking and endocytic pathways remained functional, Sptlc2-/- DC2.4 cells express reduced levels of the pattern recognition receptors Dectin-1 and TLR2 at the cell surface. Consistent with the in vitro data, compromised sphingolipid biosynthesis in mice sensitizes the animal to C. albicans infection. Sphingolipid biosynthesis is therefore critical for phagocytosis and in vivo clearance of C. albicans.

  2. Disruption of Sphingolipid Biosynthesis Blocks Phagocytosis of Candida albicans

    PubMed Central

    Schmidt, Florian I.; Freinkman, Elizaveta; Dougan, Stephanie; Dougan, Michael; Esteban, Alexandre; Maruyama, Takeshi; Strijbis, Karin; Ploegh, Hidde L.

    2015-01-01

    The ability of phagocytes to clear pathogens is an essential attribute of the innate immune response. The role of signaling lipid molecules such as phosphoinositides is well established, but the role of membrane sphingolipids in phagocytosis is largely unknown. Using a genetic approach and small molecule inhibitors, we show that phagocytosis of Candida albicans requires an intact sphingolipid biosynthetic pathway. Blockade of serine-palmitoyltransferase (SPT) and ceramide synthase-enzymes involved in sphingolipid biosynthesis- by myriocin and fumonisin B1, respectively, impaired phagocytosis by phagocytes. We used CRISPR/Cas9-mediated genome editing to generate Sptlc2-deficient DC2.4 dendritic cells, which lack serine palmitoyl transferase activity. Sptlc2-/- DC2.4 cells exhibited a stark defect in phagocytosis, were unable to bind fungal particles and failed to form a normal phagocytic cup to engulf C. albicans. Supplementing the growth media with GM1, the major ganglioside present at the cell surface, restored phagocytic activity of Sptlc2-/- DC2.4 cells. While overall membrane trafficking and endocytic pathways remained functional, Sptlc2-/- DC2.4 cells express reduced levels of the pattern recognition receptors Dectin-1 and TLR2 at the cell surface. Consistent with the in vitro data, compromised sphingolipid biosynthesis in mice sensitizes the animal to C. albicans infection. Sphingolipid biosynthesis is therefore critical for phagocytosis and in vivo clearance of C. albicans. PMID:26431038

  3. Transcriptional regulation of storage protein synthesis during dicotyledon seed filling.

    PubMed

    Verdier, Jérôme; Thompson, Richard D

    2008-09-01

    Seeds represent a major source of nutrients for human and animal livestock diets. The nutritive value of seeds is largely due to storage products which accumulate during a key phase of seed development, seed filling. In recent years, our understanding of the mechanisms regulating seed filling has advanced significantly due to the diversity of experimental approaches used. This review summarizes recent findings related to transcription factors that regulate seed storage protein accumulation. A framework for the regulation of storage protein synthesis is established which incorporates the events before, during and after seed storage protein synthesis. The transcriptional control of storage protein synthesis is accompanied by physiological and environmental controls, notably through the action of plant hormones and other intermediary metabolites. Finally, recent post-genomics analyses on different model plants have established the existence of a conserved seed filling process involving the master regulators (LEC1, LEC2, ABI3 and FUS3) but also revealed certain differences in fine regulation between plant families.

  4. Regulation of progesterone synthesis and action in bovine corpus luteum.

    PubMed

    Rekawiecki, R; Kowalik, M K; Slonina, D; Kotwica, J

    2008-12-01

    The main function of the corpus luteum (CL) is to synthesize and secrete progesterone (P4), which regulates the duration of the estrous cycle and maintains of pregnancy in many species. Both synthesis and action of this hormone is regulated by many luteotropic and luteolytic factors. Progesterone also affects its own synthesis by regulation of the activity and genes expression of crucial enzymes which control steroidogenesis. The physiological effect of P4 on luteal cells is mediated through the nuclear receptor which occurs in two specific A and B receptor isoforms and also by non-genomic pathways. The nature of non-genomic action of P4 has not been fully understood. It is possible that P4 can temporarily impair binding of oxytocin to its receptor or it can bind one of the three potential membrane receptors. It is assumed that one of these proteins, progesterone receptor membrane component 1 may be involved in regulation of CL function and it can participate in protecting bovine CL against luteolysis. This review summarize the data involving the molecular regulation of P4 synthesis, its intracellular and membrane receptor and the genomic and non-genomic action in the bovine CL.

  5. Sphingolipid base modifying enzymes in sunflower (Helianthus annuus): cloning and characterization of a C4-hydroxylase gene and a new paralogous Δ8-desaturase gene.

    PubMed

    Moreno-Pérez, Antonio J; Martínez-Force, Enrique; Garcés, Rafael; Salas, Joaquín J

    2011-05-15

    Sphingolipids are components of plant cell membranes that participate in the regulation of important physiological processes. Unlike their animal counterparts, plant sphingolipids are characterized by high levels of base C4-hydroxylation. Moreover, desaturation at the Δ8 position predominates over the Δ4 desaturation typically found in animal sphingolipids. These modifications are due to the action of C4-hydroxylases and Δ8-long chain base desaturases, and they are important for complex sphingolipids finally becoming functional. The long chain bases of sunflower sphingolipids have high levels of hydroxylated and unsaturated moieties. Here, a C4-long chain base hydroxylase was functionally characterized in sunflower plant, an enzyme that could complement the sur2Δ mutation when heterologously expressed in this yeast mutant deficient in hydroxylation. This hydroxylase was ubiquitously expressed in sunflower, with the highest levels found in the developing cotyledons. In addition, we identified a new Δ8-long base chain desaturase gene that displays strong homology to a previously reported desaturase gene. This desaturase was also expressed in yeast and was able to change the long chain base composition of the transformed host. We studied the expression of this desaturase and compared it with that of the other isoform described in sunflower. The desaturase form studied in this paper displayed higher expression levels in developing seeds.

  6. Characterization of inositol phospho-sphingolipid-phospholipase C 1 (Isc1) in Cryptococcus neoformans reveals unique biochemical features.

    PubMed

    Henry, Jennifer; Guillotte, Aimee; Luberto, Chiara; Del Poeta, Maurizio

    2011-02-18

    In this work, we biochemically characterized inositol phosphosphingolipid-phospholipase C (Isc1) from the pathogenic fungus Cryptococcus neoformans. Unlike Isc1 from other fungi and parasites which hydrolyze both fungal complex sphingolipids (IPC-PLC) and mammalian sphingomyelin (SM-PLC), C. neoformans Isc1 only exerts IPC-PLC activity. Genetic mutations thought to regulate substrate recognition in other Isc1 proteins do not restore SM-PLC activity of the cryptococcal enzyme. C. neoformans Isc1 regulates the level of complex sphingolipids and certain species of phytoceramide, especially when fungal cells are exposed to acidic stress. Since growth in acidic environments is required for C. neoformans to cause disease, this study has important implications for understanding of C. neoformans pathogenicity.

  7. Regulation of Polyhydroxybutyrate Synthesis in the Soil Bacterium Bradyrhizobium diazoefficiens

    PubMed Central

    Quelas, J. I.; Mesa, S.; Mongiardini, E. J.; Jendrossek, D.

    2016-01-01

    ABSTRACT Polyhydroxybutyrate (PHB) is a carbon and energy reserve polymer in various prokaryotic species. We determined that, when grown with mannitol as the sole carbon source, Bradyrhizobium diazoefficiens produces a homopolymer composed only of 3-hydroxybutyrate units (PHB). Conditions of oxygen limitation (such as microoxia, oxic stationary phase, and bacteroids inside legume nodules) were permissive for the synthesis of PHB, which was observed as cytoplasmic granules. To study the regulation of PHB synthesis, we generated mutations in the regulator gene phaR and the phasin genes phaP1 and phaP4. Under permissive conditions, mutation of phaR impaired PHB accumulation, and a phaP1 phaP4 double mutant produced more PHB than the wild type, which was accumulated in a single, large cytoplasmic granule. Moreover, PhaR negatively regulated the expression of phaP1 and phaP4 as well as the expression of phaA1 and phaA2 (encoding a 3-ketoacyl coenzyme A [CoA] thiolases), phaC1 and phaC2 (encoding PHB synthases), and fixK2 (encoding a cyclic AMP receptor protein [CRP]/fumarate and nitrate reductase regulator [FNR]-type transcription factor of genes for microoxic lifestyle). In addition to the depressed PHB cycling, phaR mutants accumulated more extracellular polysaccharides and promoted higher plant shoot dry weight and competitiveness for nodulation than the wild type, in contrast to the phaC1 mutant strain, which is defective in PHB synthesis. These results suggest that phaR not only regulates PHB granule formation by controlling the expression of phasins and biosynthetic enzymes but also acts as a global regulator of excess carbon allocation and symbiosis by controlling fixK2. IMPORTANCE In this work, we investigated the regulation of polyhydroxybutyrate synthesis in the soybean-nodulating bacterium Bradyrhizobium diazoefficiens and its influence in bacterial free-living and symbiotic lifestyles. We uncovered a new interplay between the synthesis of this carbon reserve

  8. Regulation of Polyhydroxybutyrate Synthesis in the Soil Bacterium Bradyrhizobium diazoefficiens.

    PubMed

    Quelas, J I; Mesa, S; Mongiardini, E J; Jendrossek, D; Lodeiro, A R

    2016-07-15

    Polyhydroxybutyrate (PHB) is a carbon and energy reserve polymer in various prokaryotic species. We determined that, when grown with mannitol as the sole carbon source, Bradyrhizobium diazoefficiens produces a homopolymer composed only of 3-hydroxybutyrate units (PHB). Conditions of oxygen limitation (such as microoxia, oxic stationary phase, and bacteroids inside legume nodules) were permissive for the synthesis of PHB, which was observed as cytoplasmic granules. To study the regulation of PHB synthesis, we generated mutations in the regulator gene phaR and the phasin genes phaP1 and phaP4 Under permissive conditions, mutation of phaR impaired PHB accumulation, and a phaP1 phaP4 double mutant produced more PHB than the wild type, which was accumulated in a single, large cytoplasmic granule. Moreover, PhaR negatively regulated the expression of phaP1 and phaP4 as well as the expression of phaA1 and phaA2 (encoding a 3-ketoacyl coenzyme A [CoA] thiolases), phaC1 and phaC2 (encoding PHB synthases), and fixK2 (encoding a cyclic AMP receptor protein [CRP]/fumarate and nitrate reductase regulator [FNR]-type transcription factor of genes for microoxic lifestyle). In addition to the depressed PHB cycling, phaR mutants accumulated more extracellular polysaccharides and promoted higher plant shoot dry weight and competitiveness for nodulation than the wild type, in contrast to the phaC1 mutant strain, which is defective in PHB synthesis. These results suggest that phaR not only regulates PHB granule formation by controlling the expression of phasins and biosynthetic enzymes but also acts as a global regulator of excess carbon allocation and symbiosis by controlling fixK2 IMPORTANCE: In this work, we investigated the regulation of polyhydroxybutyrate synthesis in the soybean-nodulating bacterium Bradyrhizobium diazoefficiens and its influence in bacterial free-living and symbiotic lifestyles. We uncovered a new interplay between the synthesis of this carbon reserve polymer

  9. Regulation of protein synthesis during sea urchin early development

    SciTech Connect

    Kelso, L.C.

    1989-01-01

    Fertilization of the sea urchin egg results in a 20-40 fold increase in the rate of protein synthesis. The masked message hypothesis proposes that mRNAs are masked or unavailable for translation in the egg. We devised an in vivo assay to test this hypothesis. Our results show that masked mRNAs limit protein synthesis in the unfertilized egg. In addition, we show that protein synthesis is also regulated at the level of translational machinery. Following fertilization is a period of rapid cell divisions. This period, known as the rapid cleavage stage, is characterized by the transient synthesis of a novel set of proteins. The synthesis of these proteins is programmed by maternal mRNAs stored in the unfertilized egg. To study the behavior of these mRNAs, we prepared a cDNA library from polysomal poly (A+) RNA from 2-hour embryos. ({sup 32}P) labeled probes, prepared from the cDNA library, were used to monitor the levels of individual mRNAs in polysomes at fertilization and during early development.

  10. Viral serine palmitoyltransferase induces metabolic switch in sphingolipid biosynthesis and is required for infection of a marine alga

    PubMed Central

    Ziv, Carmit; Malitsky, Sergey; Ben-Dor, Shifra; Wei, Yu; Zheng, Shuning; Aharoni, Asaph; Vardi, Assaf

    2016-01-01

    Marine viruses are the most abundant biological entities in the oceans shaping community structure and nutrient cycling. The interaction between the bloom-forming alga Emiliania huxleyi and its specific large dsDNA virus (EhV) is a major factor determining the fate of carbon in the ocean, thus serving as a key host-pathogen model system. The EhV genome encodes for a set of genes involved in the de novo sphingolipid biosynthesis, not reported in any viral genome to date. We combined detailed lipidomic and biochemical analyses to characterize the functional role of this virus-encoded pathway during lytic viral infection. We identified a major metabolic shift, mediated by differential substrate specificity of virus-encoded serine palmitoyltransferase, a key enzyme of sphingolipid biosynthesis. Consequently, unique viral glycosphingolipids, composed of unusual hydroxylated C17 sphingoid bases (t17:0) were highly enriched in the infected cells, and their synthesis was found to be essential for viral assembly. These findings uncover the biochemical bases of the virus-induced metabolic rewiring of the host sphingolipid biosynthesis during the chemical “arms race” in the ocean. PMID:26984500

  11. Mitochondrial Atpif1 regulates heme synthesis in developing erythroblasts

    PubMed Central

    Shah, Dhvanit I.; Takahashi-Makise, Naoko; Cooney, Jeffrey D.; Li, Liangtao; Schultz, Iman J.; Pierce, Eric L.; Narla, Anupama; Seguin, Alexandra; Hattangadi, Shilpa M.; Medlock, Amy E.; Langer, Nathaniel B.; Dailey, Tamara A.; Hurst, Slater N.; Faccenda, Danilo; Wiwczar, Jessica M.; Heggers, Spencer K.; Vogin, Guillaume; Chen, Wen; Chen, Caiyong; Campagna, Dean R.; Brugnara, Carlo; Zhou, Yi; Ebert, Benjamin L.; Danial, Nika N.; Fleming, Mark D.; Ward, Diane M.; Campanella, Michelangelo; Dailey, Harry A.; Kaplan, Jerry; Paw, Barry H.

    2012-01-01

    SUMMARY Defects in the availability of heme substrates or the catalytic activity of the terminal enzyme in heme biosynthesis, ferrochelatase (Fech), impair heme synthesis, and thus cause human congenital anemias1,2. The inter-dependent functions of regulators of mitochondrial homeostasis and enzymes responsible for heme synthesis are largely unknown. To uncover this unmet need, we utilized zebrafish genetic screens and cloned mitochondrial ATPase inhibitory factor 1 (atpif1) from a zebrafish mutant with profound anemia, pinotage (pnt tq209). We now report a direct mechanism establishing that Atpif1 regulates the catalytic efficiency of vertebrate Fech to synthesize heme. The loss of Atpif1 impairs hemoglobin synthesis in zebrafish, mouse, and human hematopoietic models as a consequence of diminished Fech activity, and elevated mitochondrial pH. To understand the relationship among mitochondrial pH, redox potential, [2Fe-2S] clusters, and Fech activity, we used (1) genetic complementation studies of Fech constructs with or without [2Fe-2S] clusters in pnt, and (2) pharmacological agents modulating mitochondrial pH and redox potential. The presence of [2Fe-2S] cluster renders vertebrate Fech vulnerable to Atpif1-regulated mitochondrial pH and redox potential perturbations. Therefore, Atpif1 deficiency reduces the efficiency of vertebrate Fech to synthesize heme, resulting in anemia. The novel mechanism of Atpif1 as a regulator of heme synthesis advances the understanding of mitochondrial heme homeostasis and red blood cell development. A deficiency of Atpif1 may contribute to important human diseases, such as congenital sideroblastic anemias and mitochondriopathies. PMID:23135403

  12. Regulation of toxin synthesis in Clostridium botulinum and Clostridium tetani.

    PubMed

    Connan, Chloé; Denève, Cécile; Mazuet, Christelle; Popoff, Michel R

    2013-12-01

    Botulinum and tetanus neurotoxins are structurally and functionally related proteins that are potent inhibitors of neuroexocytosis. Botulinum neurotoxin (BoNT) associates with non-toxic proteins (ANTPs) to form complexes of various sizes, whereas tetanus toxin (TeNT) does not form any complex. The BoNT and ANTP genes are clustered in a DNA segment called the botulinum locus, which has different genomic localization (chromosome, plasmid, phage) in the various Clostridium botulinum types and subtypes. The botulinum locus genes are organized in two polycistronic operons (ntnh-bont and ha/orfX operons) transcribed in opposite orientations. A gene called botR lying between the two operons in C. botulinum type A encodes an alternative sigma factor which regulates positively the synthesis of BoNT and ANTPs at the late exponential growth phase and beginning of the stationary phase. In Clostridium tetani, the gene located immediately upstream of tent encodes a positive regulatory protein, TetR, which is related to BotR. C. botulinum and C. tetani genomes contain several two-component systems and predicted regulatory orphan genes. In C. botulinum type A, four two-component systems have been found that positively or negatively regulate the synthesis of BoNT and ANTPs independently of BotR/A. The synthesis of neurotoxin in Clostridia seems to be under the control of complex network of regulation.

  13. Two new sphingolipids from the leaves of Piper betle L.

    PubMed

    Chen, Duo-Zhi; Xiong, Hua-Bin; Tian, Kai; Guo, Jun-Ming; Huang, Xiang-Zhong; Jiang, Zhi-Yong

    2013-09-12

    Two new sphingolipids, pipercerebrosides A (1) and B (2), were isolated from the leaves of Piper betle L. Their structures, including absolute configurations, were determined by spectroscopic analysis and chemical degradation. These two compounds did not show significant cytotoxic activity against the cancer cell lines K562 and HL-60 in a MTT assay.

  14. Calcineurin mediates homeostatic synaptic plasticity by regulating retinoic acid synthesis

    PubMed Central

    Arendt, Kristin L.; Zhang, Zhenjie; Ganesan, Subhashree; Hintze, Maik; Shin, Maggie M.; Tang, Yitai; Cho, Ahryon; Graef, Isabella A.; Chen, Lu

    2015-01-01

    Homeostatic synaptic plasticity is a form of non-Hebbian plasticity that maintains stability of the network and fidelity for information processing in response to prolonged perturbation of network and synaptic activity. Prolonged blockade of synaptic activity decreases resting Ca2+ levels in neurons, thereby inducing retinoic acid (RA) synthesis and RA-dependent homeostatic synaptic plasticity; however, the signal transduction pathway that links reduced Ca2+-levels to RA synthesis remains unknown. Here we identify the Ca2+-dependent protein phosphatase calcineurin (CaN) as a key regulator for RA synthesis and homeostatic synaptic plasticity. Prolonged inhibition of CaN activity promotes RA synthesis in neurons, and leads to increased excitatory and decreased inhibitory synaptic transmission. These effects of CaN inhibitors on synaptic transmission are blocked by pharmacological inhibitors of RA synthesis or acute genetic deletion of the RA receptor RARα. Thus, CaN, acting upstream of RA, plays a critical role in gating RA signaling pathway in response to synaptic activity. Moreover, activity blockade-induced homeostatic synaptic plasticity is absent in CaN knockout neurons, demonstrating the essential role of CaN in RA-dependent homeostatic synaptic plasticity. Interestingly, in GluA1 S831A and S845A knockin mice, CaN inhibitor- and RA-induced regulation of synaptic transmission is intact, suggesting that phosphorylation of GluA1 C-terminal serine residues S831 and S845 is not required for CaN inhibitor- or RA-induced homeostatic synaptic plasticity. Thus, our study uncovers an unforeseen role of CaN in postsynaptic signaling, and defines CaN as the Ca2+-sensing signaling molecule that mediates RA-dependent homeostatic synaptic plasticity. PMID:26443861

  15. Interictal, circulating sphingolipids in women with episodic migraine

    PubMed Central

    Mielke, Michelle M.; Dickens, Alex M.; Chatterjee, Subroto; Dash, Paul; Alexander, Guillermo; Vieira, Rebeca V.A.; Bandaru, Veera Venkata Ratnam; Dorskind, Joelle M.; Tietjen, Gretchen E.; Haughey, Norman H.

    2015-01-01

    Objective: To evaluate interictal, circulating sphingolipids in women migraineurs. Methods: In the fasting state, serum samples were obtained pain-free from 88 women with episodic migraine (EM; n = 52) and from controls (n = 36). Sphingolipids were detected and quantified by high-performance liquid chromatography coupled with tandem mass spectrometry using multiple reaction monitoring. Multivariate logistic regression was used to examine the association between serum sphingolipids and EM odds. A recursive partitioning decision tree based on the serum concentrations of 10 sphingolipids was used to determine the presence or absence of EM in a subset of participants. Results: Total ceramide (EM 6,502.9 ng/mL vs controls 10,518.5 ng/mL; p < 0.0001) and dihydroceramide (EM 39.3 ng/mL vs controls 63.1 ng/mL; p < 0.0001) levels were decreased in those with EM as compared with controls. Using multivariate logistic regression, each SD increase in total ceramide (odds ratio [OR] 0.07; 95% confidence interval [CI]: 0.02, 0.22; p < 0.001) and total dihydroceramide (OR 0.05; 95% CI: 0.01, 0.21; p < 0.001) levels was associated with more than 92% reduced odds of migraine. Although crude sphingomyelin levels were not different in EM compared with controls, after adjustments, every SD increase in the sphingomyelin species C18:0 (OR 4.28; 95% CI: 1.87, 9.81; p = 0.001) and C18:1 (OR 2.93; 95% CI: 1.55, 5.54; p = 0.001) was associated with an increased odds of migraine. Recursive portioning models correctly classified 14 of 14 randomly selected participants as EM or control. Conclusion: These results suggest that sphingolipid metabolism is altered in women with EM and that serum sphingolipid panels may have potential to differentiate EM presence or absence. Classification of evidence: This study provides Class III evidence that serum sphingolipid panels accurately distinguish women with migraine from women without migraine. PMID:26354990

  16. Sphingolipid domains in the plasma membranes of fibroblasts are not enriched with cholesterol

    SciTech Connect

    Frisz, Jessica F.; Klitzing, Haley A.; Lou, Kaiyan; Hutcheon, Ian D.; Weber, Peter K.; Zimmerberg, Joshua; Kraft, Mary L.

    2013-04-22

    The plasma membranes of mammalian cells are widely expected to contain domains that are enriched with cholesterol and sphingolipids. In this work, we have used high-resolution secondary ion mass spectrometry to directly map the distributions of isotope-labeled cholesterol and sphingolipids in the plasma membranes of intact fibroblast cells. Although acute cholesterol depletion reduced sphingolipid domain abundance, cholesterol was evenly distributed throughout the plasma membrane and was not enriched within the sphingolipid domains. As a result, we rule out favorable cholesterol-sphingolipid interactions as dictating plasma membrane organization in fibroblast cells. Because the sphingolipid domains are disrupted by drugs that depolymerize the cells actin cytoskeleton, cholesterol must instead affect the sphingolipid organization via an indirect mechanism that involves the cytoskeleton.

  17. Sphingolipid Domains in the Plasma Membranes of Fibroblasts Are Not Enriched with Cholesterol*

    PubMed Central

    Frisz, Jessica F.; Klitzing, Haley A.; Lou, Kaiyan; Hutcheon, Ian D.; Weber, Peter K.; Zimmerberg, Joshua; Kraft, Mary L.

    2013-01-01

    The plasma membranes of mammalian cells are widely expected to contain domains that are enriched with cholesterol and sphingolipids. In this work, we have used high-resolution secondary ion mass spectrometry to directly map the distributions of isotope-labeled cholesterol and sphingolipids in the plasma membranes of intact fibroblast cells. Although acute cholesterol depletion reduced sphingolipid domain abundance, cholesterol was evenly distributed throughout the plasma membrane and was not enriched within the sphingolipid domains. Thus, we rule out favorable cholesterol-sphingolipid interactions as dictating plasma membrane organization in fibroblast cells. Because the sphingolipid domains are disrupted by drugs that depolymerize the cells actin cytoskeleton, cholesterol must instead affect the sphingolipid organization via an indirect mechanism that involves the cytoskeleton. PMID:23609440

  18. Sphingolipid domains in the plasma membranes of fibroblasts are not enriched with cholesterol

    DOE PAGES

    Frisz, Jessica F.; Klitzing, Haley A.; Lou, Kaiyan; ...

    2013-04-22

    The plasma membranes of mammalian cells are widely expected to contain domains that are enriched with cholesterol and sphingolipids. In this work, we have used high-resolution secondary ion mass spectrometry to directly map the distributions of isotope-labeled cholesterol and sphingolipids in the plasma membranes of intact fibroblast cells. Although acute cholesterol depletion reduced sphingolipid domain abundance, cholesterol was evenly distributed throughout the plasma membrane and was not enriched within the sphingolipid domains. As a result, we rule out favorable cholesterol-sphingolipid interactions as dictating plasma membrane organization in fibroblast cells. Because the sphingolipid domains are disrupted by drugs that depolymerize themore » cells actin cytoskeleton, cholesterol must instead affect the sphingolipid organization via an indirect mechanism that involves the cytoskeleton.« less

  19. Dihydroceramide Desaturase Knockdown Impacts Sphingolipids and Apoptosis after Photodamage in Human Head and Neck Squamous Carcinoma Cells

    PubMed Central

    BREEN, PAUL; JOSEPH, NICHOLAS; THOMPSON, KYLE; KRAVEKA, JACQUELINE M.; GUDZ, TATYANA I.; LI, LI; RAHMANIYAN, MEHRDAD; BIELAWSKI, JACEK; PIERCE, JASON S.; VAN BUREN, ERIC; BHATTI, GAURAV; SEPAROVIC, DUSKA

    2014-01-01

    Background Dihydroceramide desaturase 1 (DES) is the enzyme responsible for converting dihydroceramide into ceramide in the de novo sphingolipid biosynthesis pathway. Dihydroceramide can inhibit ceramide channel formation to interfere with apoptosis. We have shown that following ceramide synthase knockdown, photodynamic therapy (PDT), a cancer treatment modality, is associated with decreased levels of ceramides and dihydroceramides in cells that are resistant to apoptosis. Aim Here we investigated the effect of DES knockdown on the sphingolipid profile and apoptosis in human head and neck squamous carcinoma cells after PDT with the silicon phthalocyanine Pc 4. Materials and Methods Following siRNA transfection and PDT treatment, quantitative real-time polymerase chain reaction for quantification of DES mRNA, immunoblotting for protein expression, mass spectrometry for sphingolipid analysis, spectrofluorometry for caspase 3-like (DEVDase) activity, flow cytometry for apoptosis detection, and trypan blue assay for cell viability evaluation, were performed. Results Down-regulation of DES led to a substantial increase in levels of dihydroceramides without affecting ceramide levels. PDT-induced accumulation of individual dihydroceramides and global ceramides was increased by DES knockdown. Concomitantly, mitochondrial depolarization, DEVDase activation, late-apoptosis and cell death were attenuated by DES knockdown. Early apoptosis, however, was enhanced. Conclusion Our findings support the following: (i) dihydroceramide reduces pro-apoptotic effects of ceramide; (ii) cells adapt to DES knockdown to become more sensitive to ceramide and early-apoptosis; (iii) DES is a potential molecular target for regulating apoptotic resistance to PDT. PMID:23267130

  20. Sphingolipid modulation of angiogenic factor expression in neuroblastoma

    PubMed Central

    Li, Mei-Hong; Hla, Timothy; Ferrer, Fernando

    2011-01-01

    Metabolism of sphingolipids into downstream lipid mediators followed by signaling modulates tumor microenvironment and the cancer cells to influence tumor progression. As such, sphingolipid signaling represents a novel way to modulate tumor biology. Neuroblastoma (NB), the most common extracranial solid tumor of childhood, is highly angiogenic and often displays poor prognosis. However, the role of sphingolipid mediators is not known in NB. We found that NB expresses high levels of sphingosine kinase-2 (SphK2), which is essential for the formation of sphingosine-1-phosphate (S1P). S1P induced vascular endothelial growth factor (VEGF) expression in SK-N-AS NB cells. The effect occurred at the transcriptional level. Hypoxia in combination with S1P had a synergistic effect on VEGF expression. Strong correlation was detected between S1P receptor-2 (S1P2) and VEGF mRNAs in 11 different cell lines and 17 NB tissues. Blockade of S1P2 with the selective antagonist JTE-013 significantly inhibited S1P-induced VEGF expression. Overexpression and knockdown of S1P2 in SK-N-AS cells increased or inhibited S1P-induced VEGF secretion, respectively. Interestingly, JTE-013 significantly inhibited tumor growth, VEGF mRNA expression and induced apoptosis in the NB tumor xenografts. Taken together, our data suggest that enhanced formation of sphingolipid mediator S1P in NB profoundly influences tumor microenvironment by inducing VEGF expression via S1P2. Modulation of sphingolipid signaling by inhibiting S1P2 may constitute a novel strategy to control NB. PMID:21576349

  1. Mechanisms and regulation of neurotrophin synthesis and secretion.

    PubMed

    Al-Qudah, Mohammad A; Al-Dwairi, Ahmed

    2016-10-01

    Neurotrophins are secreted proteins that are synthesized as pre-pro-neurotrophins on the rough endoplasmic reticulum, which are subsequently processed and then secreted as mature proteins. During synthesis, neurotrophins are sorted in the trans-Golgi apparatus into 2 pathways of secretion; the constitutive and the regulated pathways. Neurotrophins in the constitutive pathway are secreted cautiously without any trigger, while in the regulated pathway of secretion an external stimulus elevates the calcium concentration intracellularly leading to neurotrophin release. The regulation of sorting and secretion of neurotrophins is critical for several processes in the body, such as synaptic plasticity, neurodegenerative disorders, demyelination disease, and inflammation. The purpose of this review is to summarize the current mechanisms of neurotrophin sorting and secretion.

  2. Transcriptional regulation of decreased protein synthesis during skeletal muscle unloading

    NASA Technical Reports Server (NTRS)

    Howard, G.; Steffen, J. M.; Geoghegan, T. E.

    1989-01-01

    The regulatory role of transcriptional alterations in unloaded skeletal muscles was investigated by determining levels of total muscle RNA and mRNA fractions in soleus, gastrocnemius, and extensor digitorum longus (EDL) of rats subjected to whole-body suspension for up to 7 days. After 7 days, total RNA and mRNA contents were lower in soleus and gastrocnemius, compared with controls, but the concentrations of both RNAs per g muscle were unaltered. Alpha-actin mRNA (assessed by dot hybridization) was significantly reduced in soleus after 1, 3, and 7 days of suspension and in gastrocnemius after 3 and 7 days, but was unchanged in EDL. Protein synthesis directed by RNA extracted from soleus and EDL indicated marked alteration in mRNAs coding for several small proteins. Results suggest that altered transcription and availability of specific mRNAs contribute significantly to the regulation of protein synthesis during skeletal muscle unloading.

  3. Sphingolipids as Mediators in the Crosstalk between Microbiota and Intestinal Cells: Implications for Inflammatory Bowel Disease

    PubMed Central

    Bryan, Phillips-Farfán; Edgar Alejandro, Medina-Torres; Sara Elva, Espinosa-Padilla; Gemma, Fabrias

    2016-01-01

    Inflammatory bowel disease (IBD) describes different illnesses characterized by chronic inflammation of the gastrointestinal tract. Although the pathogenic mechanisms leading to IBD are poorly understood, immune system disturbances likely underlie its development. Sphingolipids (SLs) have been identified as important players and promising therapeutic targets to control inflammation in IBD. Interestingly, it seems that microorganisms of the normal gut microbiota and probiotics are involved in sphingolipid function. However, there is a great need to investigate the role of SLs as intermediates in the crosstalk between intestinal immunity and microorganisms. This review focuses on recent investigations that describe some mechanisms involved in the regulation of cytokine profiles by SLs. We also describe the importance of gut microbiota in providing signaling molecules that favor the communication between resident bacteria and intestinal cells. This, in turn, modulates the immune response in the bowel and likely in other peripheral organs. The potential of SLs and gut microbiota as targets or therapeutic agents for IBD is also discussed. PMID:27656050

  4. Regulation of Pseudomonas quinolone signal synthesis in Pseudomonas aeruginosa.

    PubMed

    Wade, Dana S; Calfee, M Worth; Rocha, Edson R; Ling, Elizabeth A; Engstrom, Elana; Coleman, James P; Pesci, Everett C

    2005-07-01

    Pseudomonas aeruginosa is an opportunistic pathogen that causes chronic lung infections in cystic fibrosis patients and is a major source of nosocomial infections. This bacterium controls many virulence factors by using two quorum-sensing systems, las and rhl. The las system is composed of the LasR regulator protein and its cell-to-cell signal, N-(3-oxododecanoyl) homoserine lactone, and the rhl system is composed of RhlR and the signal N-butyryl homoserine lactone. A third intercellular signal, the Pseudomonas quinolone signal (PQS; 2-heptyl-3-hydroxy-4-quinolone), also regulates numerous virulence factors. PQS synthesis requires the expression of multiple operons, one of which is pqsABCDE. Previous experiments showed that the transcription of this operon, and therefore PQS production, is negatively regulated by the rhl quorum-sensing system and positively regulated by the las quorum-sensing system and PqsR (also known as MvfR), a LysR-type transcriptional regulator protein. With the use of DNA mobility shift assays and beta-galactosidase reporter fusions, we have studied the regulation of pqsR and its relationship to pqsA, lasR, and rhlR. We show that PqsR binds the promoter of pqsA and that this binding increases dramatically in the presence of PQS, implying that PQS acts as a coinducer for PqsR. We have also mapped the transcriptional start site for pqsR and found that the transcription of pqsR is positively regulated by lasR and negatively regulated by rhlR. These results suggest that a regulatory chain occurs where pqsR is under the control of LasR and RhlR and where PqsR in turn controls pqsABCDE, which is required for the production of PQS.

  5. Sphingolipids contribute to acetic acid resistance in Zygosaccharomyces bailii.

    PubMed

    Lindahl, Lina; Genheden, Samuel; Eriksson, Leif A; Olsson, Lisbeth; Bettiga, Maurizio

    2016-04-01

    Lignocellulosic raw material plays a crucial role in the development of sustainable processes for the production of fuels and chemicals. Weak acids such as acetic acid and formic acid are troublesome inhibitors restricting efficient microbial conversion of the biomass to desired products. To improve our understanding of weak acid inhibition and to identify engineering strategies to reduce acetic acid toxicity, the highly acetic-acid-tolerant yeast Zygosaccharomyces bailii was studied. The impact of acetic acid membrane permeability on acetic acid tolerance in Z. bailii was investigated with particular focus on how the previously demonstrated high sphingolipid content in the plasma membrane influences acetic acid tolerance and membrane permeability. Through molecular dynamics simulations, we concluded that membranes with a high content of sphingolipids are thicker and more dense, increasing the free energy barrier for the permeation of acetic acid through the membrane. Z. bailii cultured with the drug myriocin, known to decrease cellular sphingo-lipid levels, exhibited significant growth inhibition in the presence of acetic acid, while growth in medium without acetic acid was unaffected by the myriocin addition. Furthermore, following an acetic acid pulse, the intracellular pH decreased more in myriocin-treated cells than in control cells. This indicates a higher inflow rate of acetic acid and confirms that the reduction in growth of cells cultured with myriocin in the medium with acetic acid was due to an increase in membrane permeability, thereby demonstrating the importance of a high fraction of sphingolipids in the membrane of Z. bailii to facilitate acetic acid resistance; a property potentially transferable to desired production organisms suffering from weak acid stress.

  6. [The first steps of chlorophyll synthesis: RNA involvement and regulation

    SciTech Connect

    Soell, D.

    1992-01-01

    Glu-tRNA[sup Glu] is synthesized from glutamate and tRNA[sup Glu] by glutamyl-tRNA synthetase (GluRS). Recent work has demonstrated that Glu-tRNA[sup Glu] has dual functions and is a precursor for protein and 5-aminolevulinate (ALA) synthesis. Current data does not provide compelling evidence for the notion that GluRS is regulated by chlorophyll precursors or in concert with the other enzymes of ALA synthesis. We have redefined the C5-pathway as a two-step route to ALA starting with Glu-tRNA[sup Glu]. Only two enzymes, Glu-tRNA reductase (GluTR) and GSA-2,1-amino-mutase (GSA-AM), are specifically involved in ALA synthesis. We have purified these enzymatic activities from Chlamydomonas and demonstrated that the two purified proteins in the presence of their cofactors NADPH and pyridoxal phosphate are sufficient for the in vitro Glu-tRNA [yields] ALA conversion. We have cloned the genes encoding GluTR. The sequences of the GluTR proteins deduced from these genes share highly conserved regions with those of bacterial origin. We havealso cloned and analyzed the gene encoding GSA-AM from Arabidopsis. As in Salmonella typhimurium, there are indications of the existence of an additional pathway for ALA formation in E. coli. To shed light on the recognition of the single tRNA[sup Glu] by the chloroplast enzymes GluTR, GluRS we characterized a chlorophyll-deficient mutant of Euglena having tRNA[sup Glu] with a point mutation in the T[Psi]C-loop. The altered tRNA supports protein but not ALA synthesis.

  7. Beyond the Cherry-Red Spot: Ocular Manifestations of Sphingolipid-mediated Neurodegenerative and Inflammatory Disorders

    PubMed Central

    Stone, Donald U.; Mandal, Nawajes A.

    2013-01-01

    Sphingolipids are a ubiquitous membrane lipid present in every cell and found most abundantly in neural tissues. Disorders such as Tay Sachs or Niemann Pick disease are the most familiar examples of dysfunction in sphingolipid metabolism and are typically associated with neurodegeneration and ocular findings such as blindness. More recently, the role of bioactive sphingolipids has been established in a multitude of cellular events, including cell survival, growth, senescence and apoptosis, inflammation, and neovascularization. We discuss our current knowledge and understanding of sphingolipid metabolism and signaling in the pathogenesis of ocular diseases. PMID:24011710

  8. Quantitative analysis of proteome and lipidome dynamics reveals functional regulation of global lipid metabolism.

    PubMed

    Casanovas, Albert; Sprenger, Richard R; Tarasov, Kirill; Ruckerbauer, David E; Hannibal-Bach, Hans Kristian; Zanghellini, Jürgen; Jensen, Ole N; Ejsing, Christer S

    2015-03-19

    Elucidating how and to what extent lipid metabolism is remodeled under changing conditions is essential for understanding cellular physiology. Here, we analyzed proteome and lipidome dynamics to investigate how regulation of lipid metabolism at the global scale supports remodeling of cellular architecture and processes during physiological adaptations in yeast. Our results reveal that activation of cardiolipin synthesis and remodeling supports mitochondrial biogenesis in the transition from fermentative to respiratory metabolism, that down-regulation of de novo sterol synthesis machinery prompts differential turnover of lipid droplet-associated triacylglycerols and sterol esters during respiratory growth, that sphingolipid metabolism is regulated in a previously unrecognized growth stage-specific manner, and that endogenous synthesis of unsaturated fatty acids constitutes an in vivo upstream activator of peroxisomal biogenesis, via the heterodimeric Oaf1/Pip2 transcription factor. Our work demonstrates the pivotal role of lipid metabolism in adaptive processes and provides a resource to investigate its regulation at the cellular level.

  9. Inhibition of GM3 Synthase Attenuates Neuropathology of Niemann-Pick Disease Type C by Affecting Sphingolipid Metabolism

    PubMed Central

    Lee, Hyun; Lee, Jong Kil; Bae, Yong Chul; Yang, Song Hyun; Okino, Nozomu; Schuchman, Edward H.; Yamashita, Tadashi; Bae, Jae-sung; Jin, Hee Kyung

    2014-01-01

    In several lysosomal storage disorders, including Niemann-Pick disease Type C (NP-C), sphingolipids, including glycosphingolipids, particularly gangliosides, are the predominant storage materials in the brain, raising the possibility that accumulation of these lipids may be involved in the NP-C neurodegenerative process. However, correlation of these accumulations and NP-C neuropathology has not been fully characterized. Here we derived NP-C mice with complete and partial deletion of the Siat9 (encoding GM3 synthase) gene in order to investigate the role of ganglioside in NP-C pathogenesis. According to our results, NPC mice with homozygotic deletion of GM3 synthase exhibited an enhanced neuropathological phenotype and died significantly earlier than NP-C mice. Notably, in contrast to complete depletion, NP-C mice with partial deletion of the GM3 synthase gene showed ameliorated NP-C neuropathology, including motor disability, demyelination, and abnormal accumulation of cholesterol and sphingolipids. These findings indicate the crucial role of GM3 synthesis in the NP-C phenotype and progression of CNS pathologic abnormality, suggesting that well-controlled inhibition of GM3 synthesis could be used as a therapeutic strategy. PMID:24599001

  10. Regulation of ribosomal protein synthesis in an Escherichia coli mutant missing ribosomal protein L1.

    PubMed Central

    Jinks-Robertson, S; Nomura, M

    1981-01-01

    In an Escherichia coli B strain missing ribosomal protein L1, the synthesis rate of L11 is 50% greater than that of other ribosomal proteins. This finding is in agreement with the previous conclusion that L1 regulates synthesis of itself and L11 and indicates that this regulation is important for maintaining the balanced synthesis of ribosomal proteins under physiological conditions. PMID:7009590

  11. Biochemistry and molecular biology of regulation of starch synthesis

    SciTech Connect

    Bloom, M.; Morell, M.; Preiss, J.

    1987-05-01

    Regulation of plant starch synthesis occurs at the level of ADPglc synthetase. 3-P-glycerate (3PGA) activates and P/sub i/ inhibits ADPG synthesis. 3PGA at high concentrations reverses P/sub i/ inhibition. Pyridoxal-P (PLP) activates the spinach leaf enzyme about 5- to 6-fold, but is not as effective as 3PGA which stimulates ADPglc synthesis 20-fold. PLP competes with 3PGA as an activator and reverses P/sub i/ inhibition thus suggesting that PLP binds at or close to the activator site. Reductive phosphopyridoxylation of the spinach leaf ADPglc synthetase with NaBH4 yields an enzyme with 5- to 6-fold higher activity in the absence of activator than the untreated enzyme when about 0.5 mol of (TH)-PLP is bound per mole of native enzyme. This modified enzyme is highly resistant to P/sub i/ inhibition in contrast to the treated enzyme. (TH)-PLP is incorporated into both 54 KD and 51 KD subunits of the enzyme. Incorporation into both peptides is inhibited by both 3PGA and P/sub i/. After incorporation of (TH)-PLP, the 51 KD subunit has been separated from the 54 KD subunit and subjected to tryptic digestion. A major radioactive peptide has been purified by HPLC and sequenced. The determined sequence was Ser-Gly-Ile-Val-Thr-Val-Ile-Lys-Asp-Ala-Leu-Ile-Pro-(Ser)- and is very similar to the deduced amino acid sequence obtained from a cDNA clone of the rice endosperm ADPglc synthetase gene. The amino acid sequence, the putative activator binding region, is situated near the C-terminus.

  12. Genes encoding Δ(8)-sphingolipid desaturase from various plants: identification, biochemical functions, and evolution.

    PubMed

    Li, Shu-Fen; Zhang, Guo-Jun; Zhang, Xue-Jin; Yuan, Jin-Hong; Deng, Chuan-Liang; Hu, Zan-Min; Gao, Wu-Jun

    2016-09-01

    ∆(8)-sphingolipid desaturase catalyzes the C8 desaturation of a long chain base, which is the characteristic structure of various complex sphingolipids. The genes of 20 ∆(8)-sphingolipid desaturases from 12 plants were identified and functionally detected by using Saccharomyces cerevisiae system to elucidate the relationship between the biochemical function and evolution of this enzyme. Results showed that the 20 genes all can encode a functional ∆(8)-sphingolipid desaturase, which catalyzes different ratios of two products, namely, 8(Z) and 8(E)-C18-phytosphingenine. The coded enzymes could be divided into two groups on the basis of biochemical functions: ∆(8)-sphingolipid desaturase with a preference for an E-isomer product and ∆(8)-sphingolipid desaturase with a preference for a Z-isomer product. The conversion rate of the latter was generally lower than that of the former. Phylogenetic analysis revealed that the 20 desaturases could also be clustered into two groups, and this grouping is consistent with that of the biochemical functions. Thus, the biochemical function of ∆(8)-sphingolipid desaturase is correlated with its evolution. The two groups of ∆(8)-sphingolipid desaturases could arise from distinct ancestors in higher plants. However, they might have initially evolved from ∆(8)-sphingolipid desaturases in lower organisms, such as yeasts, which can produce E-isomer products only. Furthermore, almost all of the transgenic yeasts harboring ∆(8)-sphingolipid desaturase genes exhibit an improvement in aluminum tolerance. Our study provided new insights into the biochemical function and evolution of ∆(8)-sphingolipid desaturases in plants.

  13. Biotin and Lipoic Acid: Synthesis, Attachment and Regulation

    PubMed Central

    Cronan, John E.

    2014-01-01

    Summary Two vitamins, biotin and lipoic acid, are essential in all three domains of life. Both coenzymes function only when covalently attached to key metabolic enzymes. There they act as “swinging arms” that shuttle intermediates between two active sites (= covalent substrate channeling) of key metabolic enzymes. Although biotin was discovered over 100 years ago and lipoic acid 60 years ago, it was not known how either coenzyme is made until recently. In Escherichia coli the synthetic pathways for both coenzymes have now been worked out for the first time. The late steps of biotin synthesis, those involved in assembling the fused rings, were well-described biochemically years ago, although recent progress has been made on the BioB reaction, the last step of the pathway in which the biotin sulfur moiety is inserted. In contrast, the early steps of biotin synthesis, assembly of the fatty acid-like “arm” of biotin were unknown. It has now been demonstrated that the arm is made by using disguised substrates to gain entry into the fatty acid synthesis pathway followed by removal of the disguise when the proper chain length is attained. The BioC methyltransferase is responsible for introducing the disguise and the BioH esterase for its removal. In contrast to biotin, which is attached to its cognate proteins as a finished molecule, lipoic acid is assembled on its cognate proteins. An octanoyl moiety is transferred from the octanoyl-ACP of fatty acid synthesis to a specific lysine residue of a cognate protein by the LipB octanoyl transferase followed by sulfur insertion at carbons C6 and C8 by the LipA lipoyl synthetase. Assembly on the cognate proteins regulates the amount of lipoic acid synthesized and thus there is no transcriptional control of the synthetic genes. In contrast transcriptional control of the biotin synthetic genes is wielded by a remarkably sophisticated, yet simple, system, exerted through BirA a dual function protein that both represses

  14. MUTANT GENES REGULATING THE INDUCIBILITY OF KYNURENINE SYNTHESIS.

    PubMed

    RIZKI, T M

    1964-05-01

    Alterations in the cellular synthesis of kynurenine in the larval fatbody of Drosophila melanogaster may be obtained by feeding the precursor tryptophan or by changing the genotype. In the wild type Ore-R strain, autofluorescent kynurenine globules normally occur in the cells in the anterior regions of the fatbody designated as regions 1, 2, and 3. When tryptophan is included in the larval diet, kynurenine will develop throughout the entire fatbody, thus extending to the cells in regions 4, 5, and 6. In the fatbodies of both the sepia mutant strain and the mutant combinations of the suppressible vermilion alleles with the suppressor gene (su(2)-s, v(1) and su(2)-s, v(2)), kynurenine is found in the cells from region 1 through region 4. This involvement of additional cells in the synthesis of kynurenine occurs under the usual culture conditions for Drosophila. When sepia larvae are fed tryptophan, kynurenine appears in all of the cells of the fatbody. However, dietary tryptophan does not induce kynurenine production in cells in regions 5 and 6 in the mutant combination su(2)-s, v(1) or su(2)-s, v(2). In the latter strains, an increase in the quantity of kynurenine in the fatbody is detected, but this increase remains limited to the same cells in which kynurenine production is found under normal feeding conditions. When the v(36f) allele is combined with the su(2)-s allele, an extremely faint autofluorescence characteristic of kynurenine is found in some of the anteriormost fat cells of regions 1 and 2. This autofluorescence becomes intensified when tryptophan is fed to su(2)-s, v(36f) larvae. The genetic control of kynurenine synthesis in the cells of the fatbody of Drosophila melanogaster has been previously demonstrated. The present observations establish genetic regulation of the ability to induce kynurenine production within a cell through the administration of the inducer tryptophan. Kynurenine production has been considered as a unit function of the cell as a

  15. Lil3 Assembles with Proteins Regulating Chlorophyll Synthesis in Barley.

    PubMed

    Mork-Jansson, Astrid; Bue, Ann Kristin; Gargano, Daniela; Furnes, Clemens; Reisinger, Veronika; Arnold, Janine; Kmiec, Karol; Eichacker, Lutz Andreas

    2015-01-01

    The light-harvesting-like (LIL) proteins are a family of membrane proteins that share a chlorophyll a/b-binding motif with the major light-harvesting antenna proteins of oxygenic photoautotrophs. LIL proteins have been associated with the regulation of tetrapyrrol biosynthesis, and plant responses to light-stress. Here, it was found in a native PAGE approach that chlorophyllide, and chlorophyllide plus geranylgeraniolpyrophosphate trigger assembly of Lil3 in three chlorine binding fluorescent protein bands, termed F1, F2, and F3. It is shown that light and chlorophyllide trigger accumulation of protochlorophyllide-oxidoreductase, and chlorophyll synthase in band F3. Chlorophyllide and chlorophyll esterified to geranylgeraniol were identified as basis of fluorescence recorded from band F3. A direct interaction between Lil3, CHS and POR was confirmed in a split ubiquitin assay. In the presence of light or chlorophyllide, geranylgeraniolpyrophosphate was shown to trigger a loss of the F3 band and accumulation of Lil3 and geranylgeranyl reductase in F1 and F2. No direct interaction between Lil3 and geranylgeraniolreductase was identified in a split ubiquitin assay; however, accumulation of chlorophyll esterified to phytol in F1 and F2 corroborated the enzymes assembly. Chlorophyll esterified to phytol and the reaction center protein psbD of photosystem II were identified to accumulate together with psb29, and APX in the fluorescent band F2. Data show that Lil3 assembles with proteins regulating chlorophyll synthesis in etioplasts from barley (Hordeum vulgare L.).

  16. Polyamines function in stress tolerance: from synthesis to regulation

    PubMed Central

    Liu, Ji-Hong; Wang, Wei; Wu, Hao; Gong, Xiaoqing; Moriguchi, Takaya

    2015-01-01

    Plants are challenged by a variety of biotic or abiotic stresses, which can affect their growth and development, productivity, and geographic distribution. In order to survive adverse environmental conditions, plants have evolved various adaptive strategies, among which is the accumulation of metabolites that play protective roles. A well-established example of the metabolites that are involved in stress responses, or stress tolerance, is the low-molecular-weight aliphatic polyamines, including putrescine, spermidine, and spermine. The critical role of polyamines in stress tolerance is suggested by several lines of evidence: firstly, the transcript levels of polyamine biosynthetic genes, as well as the activities of the corresponding enzymes, are induced by stresses; secondly, elevation of endogenous polyamine levels by exogenous supply of polyamines, or overexpression of polyamine biosynthetic genes, results in enhanced stress tolerance; and thirdly, a reduction of endogenous polyamines is accompanied by compromised stress tolerance. A number of studies have demonstrated that polyamines function in stress tolerance largely by modulating the homeostasis of reactive oxygen species (ROS) due to their direct, or indirect, roles in regulating antioxidant systems or suppressing ROS production. The transcriptional regulation of polyamine synthesis by transcription factors is also reviewed here. Meanwhile, future perspectives on polyamine research are also suggested. PMID:26528300

  17. Lil3 Assembles with Proteins Regulating Chlorophyll Synthesis in Barley

    PubMed Central

    Gargano, Daniela; Furnes, Clemens; Reisinger, Veronika; Arnold, Janine; Kmiec, Karol; Eichacker, Lutz Andreas

    2015-01-01

    The light-harvesting-like (LIL) proteins are a family of membrane proteins that share a chlorophyll a/b-binding motif with the major light-harvesting antenna proteins of oxygenic photoautotrophs. LIL proteins have been associated with the regulation of tetrapyrrol biosynthesis, and plant responses to light-stress. Here, it was found in a native PAGE approach that chlorophyllide, and chlorophyllide plus geranylgeraniolpyrophosphate trigger assembly of Lil3 in three chlorine binding fluorescent protein bands, termed F1, F2, and F3. It is shown that light and chlorophyllide trigger accumulation of protochlorophyllide-oxidoreductase, and chlorophyll synthase in band F3. Chlorophyllide and chlorophyll esterified to geranylgeraniol were identified as basis of fluorescence recorded from band F3. A direct interaction between Lil3, CHS and POR was confirmed in a split ubiquitin assay. In the presence of light or chlorophyllide, geranylgeraniolpyrophosphate was shown to trigger a loss of the F3 band and accumulation of Lil3 and geranylgeranyl reductase in F1 and F2. No direct interaction between Lil3 and geranylgeraniolreductase was identified in a split ubiquitin assay; however, accumulation of chlorophyll esterified to phytol in F1 and F2 corroborated the enzymes assembly. Chlorophyll esterified to phytol and the reaction center protein psbD of photosystem II were identified to accumulate together with psb29, and APX in the fluorescent band F2. Data show that Lil3 assembles with proteins regulating chlorophyll synthesis in etioplasts from barley (Hordeum vulgare L.). PMID:26172838

  18. Characteristics of the rat cardiac sphingolipid pool in two mitochondrial subpopulations.

    PubMed

    Monette, Jeffrey S; Gómez, Luis A; Moreau, Régis F; Bemer, Brett A; Taylor, Alan W; Hagen, Tory M

    2010-07-23

    Mitochondrial sphingolipids play a diverse role in normal cardiac function and diseases, yet a precise quantification of cardiac mitochondrial sphingolipids has never been performed. Therefore, rat heart interfibrillary mitochondria (IFM) and subsarcolemmal mitochondria (SSM) were isolated, lipids extracted, and sphingolipids quantified by LC-tandem mass spectrometry. Results showed that sphingomyelin (approximately 10,000 pmol/mg protein) was the predominant sphingolipid regardless of mitochondrial subpopulation, and measurable amounts of ceramide (approximately 70 pmol/mg protein) sphingosine, and sphinganine were also found in IFM and SSM. Both mitochondrial populations contained similar quantities of sphingolipids except for ceramide which was much higher in SSM. Analysis of sphingolipid isoforms revealed ten different sphingomyelins and six ceramides that differed from 16- to 24-carbon units in their acyl side chains. Sub-fractionation experiments further showed that sphingolipids are a constituent part of the inner mitochondrial membrane. Furthermore, inner membrane ceramide levels were 32% lower versus whole mitochondria (45 pmol/mg protein). Three ceramide isotypes (C20-, C22-, and C24-ceramide) accounted for the lower amounts. The concentrations of the ceramides present in the inner membranes of SSM and IFM differed greatly. Overall, mitochondrial sphingolipid content reflected levels seen in cardiac tissue, but the specific ceramide distribution distinguished IFM and SSM from each other.

  19. Characteristics of the Rat Cardiac Sphingolipid Pool in Two Mitochondrial Subpopulations

    PubMed Central

    Monette, Jeffrey S.; Gómez, Luis A.; Moreau, Régis F.; Bemer, Brett A.; Taylor, Alan W.; Hagen, Tory M.

    2010-01-01

    Mitochondrial sphingolipids play a diverse role in normal cardiac function and diseases, yet a precise quantification of cardiac mitochondrial sphingolipids has never been performed. Therefore, rat heart interfibrillary (IFM) and subsarcolemmal (SSM) mitochondria were isolated, lipids extracted, and sphingolipids quantified by LC-tandem mass spectrometry. Results showed that sphingomyelin (~10,000 pmols/mg protein) was the predominant sphingolipid regardless of mitochondrial subpopulation, and measurable amounts of ceramide (~70 pmols/mg protein) sphingosine, and sphinganine were also found in IFM and SSM. Both mitochondrial populations contained similar quantities of sphingolipids except for ceramide which was much higher in SSM. Analysis of sphingolipid isoforms revealed ten different sphingomyelins and six ceramides that differed from 16 to 24 carbon units in their acyl side-chains. Sub-fractionation experiments further showed that sphingolipids are a constituent part of the inner mitochondrial membrane. Furthermore, inner membrane ceramide levels were 32% lower versus whole mitochondria (45 pmols/mg protein). Three ceramide isotypes (C20-, C22-, and C24-ceramide) accounted for the lower amounts. The concentrations of the ceramides present in the inner membranes of SSM and IFM differed greatly. Overall, mitochondrial sphingolipid content reflected levels seen in cardiac tissue, but the specific ceramide distribution distinguished IFM and SSM from each other. PMID:20599536

  20. Adaptive Control Model Reveals Systematic Feedback and Key Molecules in Metabolic Pathway Regulation

    PubMed Central

    Moffitt, Richard A.; Merrill, Alfred H.; Wang, May D.

    2011-01-01

    Abstract Robust behavior in metabolic pathways resembles stabilized performance in systems under autonomous control. This suggests we can apply control theory to study existing regulation in these cellular networks. Here, we use model-reference adaptive control (MRAC) to investigate the dynamics of de novo sphingolipid synthesis regulation in a combined theoretical and experimental case study. The effects of serine palmitoyltransferase over-expression on this pathway are studied in vitro using human embryonic kidney cells. We report two key results from comparing numerical simulations with observed data. First, MRAC simulations of pathway dynamics are comparable to simulations from a standard model using mass action kinetics. The root-sum-square (RSS) between data and simulations in both cases differ by less than 5%. Second, MRAC simulations suggest systematic pathway regulation in terms of adaptive feedback from individual molecules. In response to increased metabolite levels available for de novo sphingolipid synthesis, feedback from molecules along the main artery of the pathway is regulated more frequently and with greater amplitude than from other molecules along the branches. These biological insights are consistent with current knowledge while being new that they may guide future research in sphingolipid biology. In summary, we report a novel approach to study regulation in cellular networks by applying control theory in the context of robust metabolic pathways. We do this to uncover potential insight into the dynamics of regulation and the reverse engineering of cellular networks for systems biology. This new modeling approach and the implementation routines designed for this case study may be extended to other systems. Supplementary Material is available at www.liebertonline.com/cmb. PMID:21314456

  1. Hemagglutinin Clusters in the Plasma Membrane Are Not Enriched with Cholesterol and Sphingolipids

    PubMed Central

    Wilson, Robert L.; Frisz, Jessica F.; Klitzing, Haley A.; Zimmerberg, Joshua; Weber, Peter K.; Kraft, Mary L.

    2015-01-01

    The clusters of the influenza envelope protein, hemagglutinin, within the plasma membrane are hypothesized to be enriched with cholesterol and sphingolipids. Here, we directly tested this hypothesis by using high-resolution secondary ion mass spectrometry to image the distributions of antibody-labeled hemagglutinin and isotope-labeled cholesterol and sphingolipids in the plasma membranes of fibroblast cells that stably express hemagglutinin. We found that the hemagglutinin clusters were neither enriched with cholesterol nor colocalized with sphingolipid domains. Thus, hemagglutinin clustering and localization in the plasma membrane is not controlled by cohesive interactions between hemagglutinin and liquid-ordered domains enriched with cholesterol and sphingolipids, or from specific binding interactions between hemagglutinin, cholesterol, and/or the majority of sphingolipid species in the plasma membrane. PMID:25863057

  2. Sphingolipid Organization in the Plasma Membrane and the Mechanisms That Influence It

    PubMed Central

    Kraft, Mary L.

    2017-01-01

    Sphingolipids are structural components in the plasma membranes of eukaryotic cells. Their metabolism produces bioactive signaling molecules that modulate fundamental cellular processes. The segregation of sphingolipids into distinct membrane domains is likely essential for cellular function. This review presents the early studies of sphingolipid distribution in the plasma membranes of mammalian cells that shaped the most popular current model of plasma membrane organization. The results of traditional imaging studies of sphingolipid distribution in stimulated and resting cells are described. These data are compared with recent results obtained with advanced imaging techniques, including super-resolution fluorescence detection and high-resolution secondary ion mass spectrometry (SIMS). Emphasis is placed on the new insight into the sphingolipid organization within the plasma membrane that has resulted from the direct imaging of stable isotope-labeled lipids in actual cell membranes with high-resolution SIMS. Super-resolution fluorescence techniques have recently revealed the biophysical behaviors of sphingolipids and the unhindered diffusion of cholesterol analogs in the membranes of living cells are ultimately in contrast to the prevailing hypothetical model of plasma membrane organization. High-resolution SIMS studies also conflicted with the prevailing hypothesis, showing sphingolipids are concentrated in micrometer-scale membrane domains, but cholesterol is evenly distributed within the plasma membrane. Reductions in cellular cholesterol decreased the number of sphingolipid domains in the plasma membrane, whereas disruption of the cytoskeleton eliminated them. In addition, hemagglutinin, a transmembrane protein that is thought to be a putative raft marker, did not cluster within sphingolipid-enriched regions in the plasma membrane. Thus, sphingolipid distribution in the plasma membrane is dependent on the cytoskeleton, but not on favorable interactions with

  3. Sphingolipids and mitochondrial function, lessons learned from yeast

    PubMed Central

    Spincemaille, Pieter; Cammue, Bruno P.; Thevissen, Karin

    2014-01-01

    Mitochondrial dysfunction is a hallmark of several neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease, but also of cancer, diabetes and rare diseases such as Wilson’s disease (WD) and Niemann Pick type C1 (NPC). Mitochondrial dysfunction underlying human pathologies has often been associated with an aberrant cellular sphingolipid metabolism. Sphingolipids (SLs) are important membrane constituents that also act as signaling molecules. The yeast Saccharomyces cerevisiae has been pivotal in unraveling mammalian SL metabolism, mainly due to the high degree of conservation of SL metabolic pathways. In this review we will first provide a brief overview of the major differences in SL metabolism between yeast and mammalian cells and the use of SL biosynthetic inhibitors to elucidate the contribution of specific parts of the SL metabolic pathway in response to for instance stress. Next, we will discuss recent findings in yeast SL research concerning a crucial signaling role for SLs in orchestrating mitochondrial function, and translate these findings to relevant disease settings such as WD and NPC. In summary, recent research shows that S. cerevisiae is an invaluable model to investigate SLs as signaling molecules in modulating mitochondrial function, but can also be used as a tool to further enhance our current knowledge on SLs and mitochondria in mammalian cells. PMID:28357246

  4. Haematopoietic stem cells require a highly regulated protein synthesis rate.

    PubMed

    Signer, Robert A J; Magee, Jeffrey A; Salic, Adrian; Morrison, Sean J

    2014-05-01

    Many aspects of cellular physiology remain unstudied in somatic stem cells, for example, there are almost no data on protein synthesis in any somatic stem cell. Here we set out to compare protein synthesis in haematopoietic stem cells (HSCs) and restricted haematopoietic progenitors. We found that the amount of protein synthesized per hour in HSCs in vivo was lower than in most other haematopoietic cells, even if we controlled for differences in cell cycle status or forced HSCs to undergo self-renewing divisions. Reduced ribosome function in Rpl24(Bst/+) mice further reduced protein synthesis in HSCs and impaired HSC function. Pten deletion increased protein synthesis in HSCs but also reduced HSC function. Rpl24(Bst/+) cell-autonomously rescued the effects of Pten deletion in HSCs; blocking the increase in protein synthesis, restoring HSC function, and delaying leukaemogenesis. Pten deficiency thus depletes HSCs and promotes leukaemia partly by increasing protein synthesis. Either increased or decreased protein synthesis impairs HSC function.

  5. Synthesis and characterization of TEP-EDTA-regulated bioactive hydroxyapatite

    NASA Astrophysics Data System (ADS)

    Haders, Daniel Joseph, II

    Ca2+ concentration enabled the HA crystallization process to be growth dominated, producing films composed of high crystallinity, hexagonal grains on multiple metallic substrates. TEP regulation of HA crystallization enabled the deposition of an adhesive CaTiO3 intermediate layer, and then HA in a continuous, phase sequenced process on Ti6Al4V substrates, the first such process reported in the hydrothermal HA literature. The HA film was found to be deposited by a passivating competitive growth mechanism that enabled the [0001] crystallographic orientation of hexagonal single crystals to be engineered with synthesis time. Bioactivity analysis demonstrated that films were bioactive and bone bonding. Together, these results suggest that these HA films are candidates for use on metallic orthopedic implants, namely Ti6Al4V.

  6. Regulation of Hyaluronan Synthesis in Vascular Diseases and Diabetes

    PubMed Central

    Moretto, Paola; Karousou, Evgenia; Viola, Manuela; Caon, Ilaria; Passi, Alberto; Vigetti, Davide

    2015-01-01

    Cell microenvironment has a critical role determining cell fate and modulating cell responses to injuries. Hyaluronan (HA) is a ubiquitous extracellular matrix glycosaminoglycan that can be considered a signaling molecule. In fact, interacting with several cell surface receptors can deeply shape cell behavior. In vascular biology, HA triggers smooth muscle cells (SMCs) dedifferentiation which contributes to vessel wall thickening. Furthermore, HA is able to modulate inflammation by altering the adhesive properties of endothelial cells. In hyperglycemic conditions, HA accumulates in vessels and can contribute to the diabetic complications at micro- and macrovasculature. Due to the pivotal role in favoring atherogenesis and neointima formation after injuries, HA could be a new target for cardiovascular pathologies. This review will focus on the recent findings regarding the regulation of HA synthesis in human vascular SMCs. In particular, the effects of the intracellular HA substrates availability, adenosine monophosphate-activated protein kinase (AMPK), and protein O-GlcNAcylation on the main HA synthetic enzyme (i.e., HAS2) will be discussed. PMID:25834831

  7. Is N-acetylglutamate a short-term regulator of urea synthesis?

    PubMed Central

    Lund, P; Wiggins, D

    1984-01-01

    A method is described for determining N-acetylglutamate as glutamate. N-Acetylglutamate content of hepatocytes from 48 h-starved rats is high. It shows no parallelism with rates of urea synthesis from glutamine. We question its accepted function as a short-term regulator of urea synthesis. PMID:6721845

  8. Sphingosine-1-phosphate Phosphatase 2 Regulates Pancreatic Islet β-Cell Endoplasmic Reticulum Stress and Proliferation.

    PubMed

    Taguchi, Yoshimitsu; Allende, Maria L; Mizukami, Hiroki; Cook, Emily K; Gavrilova, Oksana; Tuymetova, Galina; Clarke, Benjamin A; Chen, Weiping; Olivera, Ana; Proia, Richard L

    2016-06-03

    Sphingosine-1-phosphate (S1P) is a sphingolipid metabolite that regulates basic cell functions through metabolic and signaling pathways. Intracellular metabolism of S1P is controlled, in part, by two homologous S1P phosphatases (SPPases), 1 and 2, which are encoded by the Sgpp1 and Sgpp2 genes, respectively. SPPase activity is needed for efficient recycling of sphingosine into the sphingolipid synthesis pathway. SPPase 1 is important for skin homeostasis, but little is known about the functional role of SPPase 2. To identify the functions of SPPase 2 in vivo, we studied mice with the Sgpp2 gene deleted. In contrast to Sgpp1(-/-) mice, Sgpp2(-/-) mice had normal skin and were viable into adulthood. Unexpectedly, WT mice expressed Sgpp2 mRNA at high levels in pancreatic islets when compared with other tissues. Sgpp2(-/-) mice had normal pancreatic islet size; however, they exhibited defective adaptive β-cell proliferation that was demonstrated after treatment with either a high-fat diet or the β-cell-specific toxin, streptozotocin. Importantly, β-cells from untreated Sgpp2(-/-) mice showed significantly increased expression of proteins characteristic of the endoplasmic reticulum stress response compared with β-cells from WT mice, indicating a basal islet defect. Our results show that Sgpp2 deletion causes β-cell endoplasmic reticulum stress, which is a known cause of β-cell dysfunction, and reveal a juncture in the sphingolipid recycling pathway that could impact the development of diabetes.

  9. Quantitative profiling of sphingolipids in wild Cordyceps and its mycelia by using UHPLC-MS.

    PubMed

    Mi, Jia-Ning; Wang, Jing-Rong; Jiang, Zhi-Hong

    2016-02-12

    In the present study, 101 sphingolipids in wild Cordyceps and its five mycelia were quantitatively profiled by using a fully validated UHPLC-MS method. The results revealed that a general rank order for the abundance of different classes of sphingolipids in wild Cordyceps and its mycelia is sphingoid bases/ceramides > phosphosphingolipids > glycosphingolipids. However, remarkable sphingolipid differences between wild Cordyceps and its mycelia were observed. One is that sphingoid base is the dominant sphingolipid in wild Cordyceps, whereas ceramide is the major sphingolipid in mycelia. Another difference is that the abundance of sphingomyelins in wild Cordyceps is almost 10-folds higher than those in most mycelia. The third one is that mycelia contain more inositol phosphorylceramides and glycosphingolipids than wild Cordyceps. Multivariate analysis was further employed to visualize the difference among wild Cordyceps and different mycelia, leading to the identification of respective sphingolipids as potential chemical markers for the differentiation of wild Cordyceps and its related mycelia. This study represents the first report on the quantitative profiling of sphingolipids in wild Cordyceps and its related mycelia, which provided comprehensive chemical evidence for the quality control and rational utilization of wild Cordyceps and its mycelia.

  10. Quantitative profiling of sphingolipids in wild Cordyceps and its mycelia by using UHPLC-MS

    PubMed Central

    Mi, Jia-Ning; Wang, Jing-Rong; Jiang, Zhi-Hong

    2016-01-01

    In the present study, 101 sphingolipids in wild Cordyceps and its five mycelia were quantitatively profiled by using a fully validated UHPLC-MS method. The results revealed that a general rank order for the abundance of different classes of sphingolipids in wild Cordyceps and its mycelia is sphingoid bases/ceramides > phosphosphingolipids > glycosphingolipids. However, remarkable sphingolipid differences between wild Cordyceps and its mycelia were observed. One is that sphingoid base is the dominant sphingolipid in wild Cordyceps, whereas ceramide is the major sphingolipid in mycelia. Another difference is that the abundance of sphingomyelins in wild Cordyceps is almost 10-folds higher than those in most mycelia. The third one is that mycelia contain more inositol phosphorylceramides and glycosphingolipids than wild Cordyceps. Multivariate analysis was further employed to visualize the difference among wild Cordyceps and different mycelia, leading to the identification of respective sphingolipids as potential chemical markers for the differentiation of wild Cordyceps and its related mycelia. This study represents the first report on the quantitative profiling of sphingolipids in wild Cordyceps and its related mycelia, which provided comprehensive chemical evidence for the quality control and rational utilization of wild Cordyceps and its mycelia. PMID:26868933

  11. Tackling the biophysical properties of sphingolipids to decipher their biological roles.

    PubMed

    Carreira, Ana C; Ventura, Ana E; Varela, Ana R P; Silva, Liana C

    2015-06-01

    From the most simple sphingoid bases to their complex glycosylated derivatives, several sphingolipid species were shown to have a role in fundamental cellular events and/or disease. Increasing evidence places lipid-lipid interactions and membrane structural alterations as central mechanisms underlying the action of these lipids. Understanding how these molecules exert their biological roles by studying their impact in the physical properties and organization of membranes is currently one of the main challenges in sphingolipid research. Herein, we review the progress in the state-of-the-art on the biophysical properties of sphingolipid-containing membranes, focusing on sphingosine, ceramides, and glycosphingolipids.

  12. Structure and function of sphingosine-1-phosphate lyase, a key enzyme of sphingolipid metabolism.

    PubMed

    Bourquin, Florence; Riezman, Howard; Capitani, Guido; Grütter, Markus G

    2010-08-11

    Sphingosine-1-phosphate lyase (SPL), a key enzyme of sphingolipid metabolism, catalyzes the irreversible degradation of sphingoid base phosphates. Its main substrate sphingosine-1-phosphate (S1P) acts both extracellularly, by binding G protein-coupled receptors of the lysophospholipid receptor family, and inside the cell, as a second messenger. There, S1P takes part in regulating various cellular processes and its levels are tightly regulated. SPL is a pivotal enzyme regulating S1P intracellular concentrations and a promising drug target for the design of immunosuppressants. We structurally and functionally characterized yeast SPL (Dpl1p) and its first prokaryotic homolog, from Symbiobacterium thermophilum. The Dpl1p structure served as a basis for a very reliable model of Homo sapiens SPL. The above results, together with in vitro and in vivo studies of SPL mutants, reveal which residues are involved in activity and substrate binding and pave the way to studies aimed at controlling the activity of this pivotal enzyme.

  13. Sphingolipid trafficking and purification in Chlamydia trachomatis-infected cells

    PubMed Central

    2012-01-01

    Chlamydia trachomatis is an obligate intracellular human pathogen, which lacks a system that allows genetic manipulation. Therefore, chlamydial researchers must manipulate the host cell to better understand chlamydial biology. Host-derived lipid acquisition is critical for chlamydial survival within the host. Hence, the ability to track and purify sphingolipids in/from chlamydial infected cells has become an integral part of pivotal studies in chlamydial biology. This Unit outlines protocols that provide details about labeling eukaryotic cells with exogenous lipids to examine Golgi-derived lipid trafficking to the chlamydial inclusion and then performing imaging studies or lipid extractions for quantification. Details are provided to allow these protocols to be applied to subconfluent, polarized or siRNA knockdown cells. In addition, one will find important experimental design considerations and techniques. These methods are powerful tools to aid in the understanding of mechanisms which allow C. trachomatis to manipulate and usurp host cell trafficking pathways. PMID:23184593

  14. An introduction to sphingolipid metabolism and analysis by new technologies.

    PubMed

    Chen, Yanfeng; Liu, Ying; Sullards, M Cameron; Merrill, Alfred H

    2010-12-01

    Sphingolipids (SP) are a complex class of molecules found in essentially all eukaryotes and some prokaryotes and viruses where they influence membrane structure, intracellular signaling, and interactions with the extracellular environment. Because of the combinatorial nature of their biosynthesis, there are thousands of SP subspecies varying in the lipid backbones and complex phospho- and glycoheadgroups. Therefore, comprehensive or "sphingolipidomic" analyses (structure-specific, quantitative analyses of all SP, or at least all members of a critical subset) are needed to know which and how much of these subspecies are present in a system as a step toward understanding their functions. Mass spectrometry and related novel techniques are able to quantify a small fraction, but nonetheless a substantial number, of SP and are beginning to provide information about their localization. This review summarizes the basic metabolism of SP and state-of-art mass spectrometric techniques that are producing insights into SP structure, metabolism, functions, and some of the dysfunctions of relevance to neuromedicine.

  15. VCP and ATL1 regulate endoplasmic reticulum and protein synthesis for dendritic spine formation.

    PubMed

    Shih, Yu-Tzu; Hsueh, Yi-Ping

    2016-03-17

    Imbalanced protein homeostasis, such as excessive protein synthesis and protein aggregation, is a pathogenic hallmark of a range of neurological disorders. Here, using expression of mutant proteins, a knockdown approach and disease mutation knockin mice, we show that VCP (valosin-containing protein), together with its cofactor P47 and the endoplasmic reticulum (ER) morphology regulator ATL1 (Atlastin-1), regulates tubular ER formation and influences the efficiency of protein synthesis to control dendritic spine formation in neurons. Strengthening the significance of protein synthesis in dendritic spinogenesis, the translation blocker cyclohexamide and the mTOR inhibitor rapamycin reduce dendritic spine density, while a leucine supplement that increases protein synthesis ameliorates the dendritic spine defects caused by Vcp and Atl1 deficiencies. Because VCP and ATL1 are the causative genes of several neurodegenerative and neurodevelopmental disorders, we suggest that impaired ER formation and inefficient protein synthesis are significant in the pathogenesis of multiple neurological disorders.

  16. VCP and ATL1 regulate endoplasmic reticulum and protein synthesis for dendritic spine formation

    PubMed Central

    Shih, Yu-Tzu; Hsueh, Yi-Ping

    2016-01-01

    Imbalanced protein homeostasis, such as excessive protein synthesis and protein aggregation, is a pathogenic hallmark of a range of neurological disorders. Here, using expression of mutant proteins, a knockdown approach and disease mutation knockin mice, we show that VCP (valosin-containing protein), together with its cofactor P47 and the endoplasmic reticulum (ER) morphology regulator ATL1 (Atlastin-1), regulates tubular ER formation and influences the efficiency of protein synthesis to control dendritic spine formation in neurons. Strengthening the significance of protein synthesis in dendritic spinogenesis, the translation blocker cyclohexamide and the mTOR inhibitor rapamycin reduce dendritic spine density, while a leucine supplement that increases protein synthesis ameliorates the dendritic spine defects caused by Vcp and Atl1 deficiencies. Because VCP and ATL1 are the causative genes of several neurodegenerative and neurodevelopmental disorders, we suggest that impaired ER formation and inefficient protein synthesis are significant in the pathogenesis of multiple neurological disorders. PMID:26984393

  17. Plasma sphingolipids in HIV-associated chronic obstructive pulmonary disease

    PubMed Central

    Hodgson, Shane; Griffin, Timothy J; Reilly, Cavan; Harvey, Stephen; Witthuhn, Bruce A; Sandri, Brian J; Wendt, Chris H

    2017-01-01

    Introduction Chronic obstructive pulmonary disease (COPD) is a significant cause of morbidity in persons living with HIV (PLWH) and HIV appears to uniquely cause COPD, independent of smoking. The mechanisms by which HIV leads to COPD are not clear. The objective of this study was to identify metabolomic biomarkers and potential mechanistic pathways of HIV-associated COPD (HIV-COPD). Methods We performed case–control metabolite profiling via mass spectrometry in plasma from 38 individuals with HIV-COPD (cases), comparing to matched controls with/without HIV and with/without COPD. Untargeted metabolites of interest were identified with liquid chromatography with mass spectrometry (LC-MS/mass spectrometry (MS)), and targeted metabolomics for tryptophan (Trp) and kynurenine (Kyn) were measured by selective reaction monitoring (SRM) with LC-MS/MS. We used mixed-effects models to compare metabolite concentrations in cases compared with controls while controlling for relevant biological variables. Results We identified 1689 analytes associated with HIV-COPD at a false discovery rate (FDR) of 10%. In PLWH, we identified 263 analytes (10% FDR) between those with and without COPD. LC MS/MS identified Trp and 17 lipids, including sphingolipids and diacylglycerol. After adjusting for relevant covariates, the Kyn/Trp ratio measured by SRM was significantly higher in PLWH (p=0.022), but was not associated with COPD status (p=0.95). Conclusions There is a unique metabolite profile in HIV-COPD that includes sphingolipids. Trp metabolism is increased in HIV, but does not appear to independently contribute to HIV-COPD. Trial registration numbers NCT01810289, NCT01797367, NCT00608764.

  18. Jasmonate Hormone: Regulating Synthesis of Reduced Carbon Compounds in Plants

    SciTech Connect

    Browse, John

    2016-05-13

    Our original interest in understanding the role of jasmonate (JA) in regulating the final stages of stamen and pollen development led to our discovery of the JAZ repressors, and the molecular mechanism of JA action is now a second important focus of our research. The specific goals for this grant period are to: 1. Investigate the generation and clearance of the hormone with emphasis on the regulation of the OPR3 enzyme and the hydrolysis of JA-Ile. 2. Use dominant-negative and overexpression constructs to explore the role of the MYC5 transcription factor in initiating and regulating JA responses. 3. Investigate specific JAZ protein interactions that will help us to recognize and understand the extended network of processes, such as sulfur nutrition, that interface with JA signaling. The COI1 F-Box protein is a JA-Ile coreceptor and coi1 mutant plants lack JA responses. We have tested the possibility that sites of JA action can be probed by using tissue-specific promoters to drive expression of a COI1-YFP fusion protein in coi1 mutant plants deficient in stamen and pollen function. When we expressed COI1 behind a filament-specific promoter (from the DAD1 gene), filament elongation was restored but not anther dehiscence or pollen function. Three tapetum specific promoters, all failed to restore any of these three functions but, unexpectedly, a promoter active in the stomium and epidermal cells, restored both pollen function and anther dehiscence. Most importantly, our results demonstrate the power of promoter::COI1-YFP constructs in revealing the primary sites of JA-regulated gene expression that control developmental and other responses in neighboring tissues. We now plan to use this new tool to test current hypotheses about JA action in other organs of the plant. The MYC2, MYC3, and MYC4 proteins are the primary transcription factors initiating defense and root growth responses to JA signaling. However, transgenic plants overexpressing these proteins do not show

  19. Comprehensive Plasma Metabolomic Analyses of Atherosclerotic Progression Reveal Alterations in Glycerophospholipid and Sphingolipid Metabolism in Apolipoprotein E-deficient Mice

    PubMed Central

    Dang, Vi T.; Huang, Aric; Zhong, Lexy H.; Shi, Yuanyuan; Werstuck, Geoff H.

    2016-01-01

    Atherosclerosis is the major underlying cause of most cardiovascular diseases. Despite recent advances, the molecular mechanisms underlying the pathophysiology of atherogenesis are not clear. In this study, comprehensive plasma metabolomics were used to investigate early-stage atherosclerotic development and progression in chow-fed apolipoprotein E-deficient mice at 5, 10 and 15 weeks of age. Comprehensive plasma metabolomic profiles, based on 4365 detected metabolite features, differentiate atherosclerosis-prone from atherosclerosis-resistant models. Metabolites in the sphingomyelin pathway were significantly altered prior to detectable lesion formation and at all subsequent time-points. The cytidine diphosphate-diacylglycerol pathway was up-regulated during stage I of atherosclerosis, while metabolites in the phosphatidylethanolamine and glycosphingolipid pathways were augmented in mice with stage II lesions. These pathways, involving glycerophospholipid and sphingolipid metabolism, were also significantly affected during the course of atherosclerotic progression. Our findings suggest that distinct plasma metabolomic profiles can differentiate the different stages of atherosclerotic progression. This study reveals that alteration of specific, previously unreported pathways of glycerophospholipid and sphingolipid metabolism are associated with atherosclerosis. The clear difference in the level of several metabolites supports the use of plasma lipid profiling as a diagnostic tool of atherogenesis. PMID:27721472

  20. Sphingolipids, Lipid Rafts, and Giardial Encystation: The Show Must Go On.

    PubMed

    Mendez, Tavis L; De Chatterjee, Atasi; Duarte, Trevor; De Leon, Joaquin; Robles-Martinez, Leobarda; Das, Siddhartha

    2015-09-01

    Sphingolipids are sphingosine-based phospholipids, which are present in the plasma and endomembranes of many eukaryotic cells. These lipids are involved in various cellular functions, including cell growth, differentiation, and apoptosis. In addition, sphingolipid and cholesterol-enriched membrane microdomains (also called "lipid rafts") contain a set of proteins and lipids, which take part in the signaling process in response to intra- or extracellular stimuli. Recent findings suggest that sphingolipids, especially glucosylceramide, play a critical role in inducing encystation and maintaining the cyst viability in Giardia. Similarly, the assembly/disassembly of lipid rafts modulates the encystation and cyst production of this ubiquitous enteric parasite. In this review article, we discuss the overall progress in the field and examine whether sphingolipids and lipid rafts can be used as novel targets for designing therapies to control infection by Giardia, which is rampant in developing countries, where children are especially vulnerable.

  1. Trafficking and Functions of Bioactive Sphingolipids: Lessons from Cells and Model Membranes

    PubMed Central

    Zhou, Kecheng; Blom, Tomas

    2015-01-01

    Ceramide and sphingosine and their phosphorylated counterparts are recognized as “bioactive sphingolipids” and modulate membrane integrity, the activity of enzymes, or act as ligands of G protein-coupled receptors. The subcellular distribution of the bioactive sphingolipids is central to their function as the same lipid can mediate diametrically opposite effects depending on its location. To ensure that these lipids are present in the right amount and in the appropriate organelles, cells employ selective lipid transport and compartmentalize sphingolipid-metabolizing enzymes to characteristic subcellular sites. Our knowledge of key mechanisms involved in sphingolipid signaling and trafficking has increased substantially in the past decades—thanks to advances in biochemical and cell biological methods. In this review, we focus on the bioactive sphingolipids and discuss how the combination of studies in cells and in model membranes have contributed to our understanding of how they behave and function in living organisms. PMID:26715852

  2. Changes in membrane sphingolipid composition modulate dynamics and adhesion of integrin nanoclusters

    PubMed Central

    Eich, Christina; Manzo, Carlo; Keijzer, Sandra de; Bakker, Gert-Jan; Reinieren-Beeren, Inge; García-Parajo, Maria F.; Cambi, Alessandra

    2016-01-01

    Sphingolipids are essential constituents of the plasma membrane (PM) and play an important role in signal transduction by modulating clustering and dynamics of membrane receptors. Changes in lipid composition are therefore likely to influence receptor organisation and function, but how this precisely occurs is difficult to address given the intricacy of the PM lipid-network. Here, we combined biochemical assays and single molecule dynamic approaches to demonstrate that the local lipid environment regulates adhesion of integrin receptors by impacting on their lateral mobility. Induction of sphingomyelinase (SMase) activity reduced sphingomyelin (SM) levels by conversion to ceramide (Cer), resulting in impaired integrin adhesion and reduced integrin mobility. Dual-colour imaging of cortical actin in combination with single molecule tracking of integrins showed that this reduced mobility results from increased coupling to the actin cytoskeleton brought about by Cer formation. As such, our data emphasizes a critical role for the PM local lipid composition in regulating the lateral mobility of integrins and their ability to dynamically increase receptor density for efficient ligand binding in the process of cell adhesion. PMID:26869100

  3. Sphingolipids Are Dual Specific Drug Targets for the Management of Pulmonary Infections: Perspective

    PubMed Central

    Sharma, Lalita; Prakash, Hridayesh

    2017-01-01

    Sphingolipids are the major constituent of the mucus secreted by the cells of epithelial linings of lungs where they maintain the barrier functions and prevent microbial invasion. Sphingolipids are interconvertible, and their primary and secondary metabolites have both structural and functional roles. Out of several sphingolipid metabolites, sphingosine-1 phosphate (S1P) and ceramide are central molecules and decisive for sphingolipid signaling. These are produced by enzymatic activity of sphingosine kinase-1 (SK-1) upon the challenge with either biological or physiological stresses. S1P and ceramide rheostat are important for the progression of various pathologies, which are manifested by inflammatory cascade. S1P is a well-established secondary messenger and associated with various neuronal, metabolic, and inflammatory diseases other than respiratory infections such as Chlamydia pneumoniae, Streptococcus pneumoniae, and Mycobacterium tuberculosis. These pathogens are known to exploit sphingolipid metabolism for their opportunistic survival. Decreased sphingosine kinase activity/S1P content in the lung and peripheral blood of tuberculosis patients clearly indicated a dysregulation of sphingolipid metabolism during infection and suggest that sphingolipid metabolism is important for management of infection by the host. Our previous study has demonstrated that gain of SK-1 activity is important for the maturation of phagolysosomal compartment, innate activation of macrophages, and subsequent control of mycobacterial replication/growth in macrophages. Furthermore, S1P-mediated amelioration of lung pathology and disease severity in TB patients is believed to be mediated by the selective activation or rearrangement of various S1P receptors (S1PR) particularly S1PR2, which has been effective in controlling respiratory fungal pathogens. Therefore, such specificity of S1P–S1PR would be paramount for triggering inflammatory events, subsequent activation, and fostering

  4. Regulation of phospholipid synthesis in phosphatidylserine synthase-deficient (chol) mutants of Saccharomyces cerevisiae.

    PubMed Central

    Letts, V A; Henry, S A

    1985-01-01

    chol mutants of Saccharomyces cerevisiae are deficient in the synthesis of the phospholipid phosphatidylserine owing to lowered activity of the membrane-associated enzyme phosphatidylserine synthase. chol mutants are auxotrophic for ethanolamine or choline and, in the absence of these supplements, cannot synthesize phosphatidylethanolamine or phosphatidylcholine (PC). We exploited these characteristics of the chol mutants to examine the regulation of phospholipid metabolism in S. cerevisiae. Macromolecular synthesis and phospholipid metabolism were examined in chol cells starved for ethanolamine. As expected, when chol mutants were starved for ethanolamine, the rates of synthesis of the phospholipids phosphatidylethanolamine and PC declined rapidly. Surprisingly, however, coupled to the decline in PC biosynthesis was a simultaneous decrease in the overall rate of phospholipid synthesis. In particular, the rate of synthesis of phosphatidylinositol decreased in parallel with the decline in PC biosynthesis. The results obtained suggest that the slowing of PC biosynthesis in ethanolamine-starved chol cells leads to a coordinated decrease in the synthesis of all phospholipids. However, under conditions of ethanolamine deprivation in chol cells, the cytoplasmic enzyme inositol-1-phosphate synthase could not be repressed by exogenous inositol, and the endogenous synthesis of the phospholipid precursor inositol appeared to be elevated. The implications of these findings with respect to the coordinated regulation of phospholipid synthesis are discussed. Images PMID:2991194

  5. Lewis lung carcinoma regulation of mechanical stretch-induced protein synthesis in cultured myotubes.

    PubMed

    Gao, Song; Carson, James A

    2016-01-01

    Mechanical stretch can activate muscle and myotube protein synthesis through mammalian target of rapamycin complex 1 (mTORC1) signaling. While it has been established that tumor-derived cachectic factors can induce myotube wasting, the effect of this catabolic environment on myotube mechanical signaling has not been determined. We investigated whether media containing cachectic factors derived from Lewis lung carcinoma (LLC) can regulate the stretch induction of myotube protein synthesis. C2C12 myotubes preincubated in control or LLC-derived media were chronically stretched. Protein synthesis regulation by anabolic and catabolic signaling was then examined. In the control condition, stretch increased mTORC1 activity and protein synthesis. The LLC treatment decreased basal mTORC1 activity and protein synthesis and attenuated the stretch induction of protein synthesis. LLC media increased STAT3 and AMP-activated protein kinase phosphorylation in myotubes, independent of stretch. Both stretch and LLC independently increased ERK1/2, p38, and NF-κB phosphorylation. In LLC-treated myotubes, the inhibition of ERK1/2 and p38 rescued the stretch induction of protein synthesis. Interestingly, either leukemia inhibitory factor or glycoprotein 130 antibody administration caused further inhibition of mTORC1 signaling and protein synthesis in stretched myotubes. AMP-activated protein kinase inhibition increased basal mTORC1 signaling activity and protein synthesis in LLC-treated myotubes, but did not restore the stretch induction of protein synthesis. These results demonstrate that LLC-derived cachectic factors can dissociate stretch-induced signaling from protein synthesis through ERK1/2 and p38 signaling, and that glycoprotein 130 signaling is associated with the basal stretch response in myotubes.

  6. Proteomic and functional analyses reveal MAPK1 regulates milk protein synthesis.

    PubMed

    Lu, Li-Min; Li, Qing-Zhang; Huang, Jian-Guo; Gao, Xue-Jun

    2012-12-27

    L-Lysine (L-Lys) is an essential amino acid that plays fundamental roles in protein synthesis. Many nuclear phosphorylated proteins such as Stat5 and mTOR regulate milk protein synthesis. However, the details of milk protein synthesis control at the transcript and translational levels are not well known. In this current study, a two-dimensional gel electrophoresis (2-DE)/MS-based proteomic technology was used to identify phosphoproteins responsible for milk protein synthesis in dairy cow mammary epithelial cells (DCMECs). The effect of L-Lys on DCMECs was analyzed by CASY technology and reversed phase high performance liquid chromatography (RP-HPLC). The results showed that cell proliferation ability and β-casein expression were enhanced in DCMECs treated with L-Lys. By phosphoproteomics analysis, six proteins, including MAPK1, were identified up-expressed in DCMECs treated with 1.2 mM L-Lys for 24 h, and were verified by quantitative real-time PCR (qRT-PCR) and western blot. Overexpression and siRNA inhibition of MAPK1 experiments showed that MAPK1 upregulated milk protein synthesis through Stat5 and mTOR pathway. These findings that MAPK1 involves in regulation of milk synthesis shed new insights for understanding the mechanisms of milk protein synthesis.

  7. A Gibberellin-Mediated DELLA-NAC Signaling Cascade Regulates Cellulose Synthesis in Rice[OPEN

    PubMed Central

    Huang, Debao; Wang, Shaogan; Zhang, Baocai; Shang-Guan, Keke; Shi, Yanyun; Zhang, Dongmei; Liu, Xiangling; Wu, Kun; Xu, Zuopeng; Fu, Xiangdong; Zhou, Yihua

    2015-01-01

    Cellulose, which can be converted into numerous industrial products, has important impacts on the global economy. It has long been known that cellulose synthesis in plants is tightly regulated by various phytohormones. However, the underlying mechanism of cellulose synthesis regulation remains elusive. Here, we show that in rice (Oryza sativa), gibberellin (GA) signals promote cellulose synthesis by relieving the interaction between SLENDER RICE1 (SLR1), a DELLA repressor of GA signaling, and NACs, the top-layer transcription factors for secondary wall formation. Mutations in GA-related genes and physiological treatments altered the transcription of CELLULOSE SYNTHASE genes (CESAs) and the cellulose level. Multiple experiments demonstrated that transcription factors NAC29/31 and MYB61 are CESA regulators in rice; NAC29/31 directly regulates MYB61, which in turn activates CESA expression. This hierarchical regulation pathway is blocked by SLR1-NAC29/31 interactions. Based on the results of anatomical analysis and GA content examination in developing rice internodes, this signaling cascade was found to be modulated by varied endogenous GA levels and to be required for internode development. Genetic and gene expression analyses were further performed in Arabidopsis thaliana GA-related mutants. Altogether, our findings reveal a conserved mechanism by which GA regulates secondary wall cellulose synthesis in land plants and provide a strategy for manipulating cellulose production and plant growth. PMID:26002868

  8. Sphingolipids, Transcription Factors, and Conserved Toolkit Genes: Developmental Plasticity in the Ant Cardiocondyla obscurior

    PubMed Central

    Schrader, Lukas; Simola, Daniel F.; Heinze, Jürgen; Oettler, Jan

    2015-01-01

    Developmental plasticity allows for the remarkable morphological specialization of individuals into castes in eusocial species of Hymenoptera. Developmental trajectories that lead to alternative caste fates are typically determined by specific environmental stimuli that induce larvae to express and maintain distinct gene expression patterns. Although most eusocial species express two castes, queens and workers, the ant Cardiocondyla obscurior expresses diphenic females and males; this provides a unique system with four discrete phenotypes to study the genomic basis of developmental plasticity in ants. We sequenced and analyzed the transcriptomes of 28 individual C. obscurior larvae of known developmental trajectory, providing the first in-depth analysis of gene expression in eusocial insect larvae. Clustering and transcription factor binding site analyses revealed that different transcription factors and functionally distinct sets of genes are recruited during larval development to induce the four alternative trajectories. In particular, we found complex patterns of gene regulation pertaining to sphingolipid metabolism, a conserved molecular pathway involved in development, obesity, and aging. PMID:25725431

  9. The role of sphingolipid signalling in diabetes-associated pathologies (Review)

    PubMed Central

    Ng, Mei Li; Wadham, Carol; Sukocheva, Olga A.

    2017-01-01

    Sphingosine kinase (SphK) is an important signalling enzyme that catalyses the phosphorylation of sphingosine (Sph) to form sphingosine-1-phosphate (S1P). The multifunctional lipid, S1P binds to a family of five G protein-coupled receptors (GPCRs). As an intracellular second messenger, S1P activates key signalling cascades responsible for the maintenance of sphingolipid metabolism, and has been implicated in the progression of cancer, and the development of other inflammatory and metabolic diseases. SphK and S1P are critical molecules involved in the regulation of various cellular metabolic processes, such as cell proliferation, survival, apoptosis, adhesion and migration. There is strong evidence supporting the critical roles of SphK and S1P in the progression of diabetes mellitus, including insulin sensitivity and insulin secretion, pancreatic β-cell apoptosis, and the development of diabetic inflammatory state. In this review, we summarise the current state of knowledge for SphK/S1P signalling effects, associated with the development of insulin resistance, pancreatic β-cell death and the vascular complications of diabetes mellitus. PMID:28075451

  10. Syndecan-2 regulates melanin synthesis via protein kinase C βII-mediated tyrosinase activation.

    PubMed

    Jung, Hyejung; Chung, Heesung; Chang, Sung Eun; Choi, Sora; Han, Inn-Oc; Kang, Duk-Hee; Oh, Eok-Soo

    2014-05-01

    Syndecan-2, a transmembrane heparan sulfate proteoglycan that is highly expressed in melanoma cells, regulates melanoma cell functions (e.g. migration). Since melanoma is a malignant tumor of melanocytes, which largely function to synthesize melanin, we investigated the possible involvement of syndecan-2 in melanogenesis. Syndecan-2 expression was increased in human skin melanoma tissues compared with normal skin. In both mouse and human melanoma cells, siRNA-mediated knockdown of syndecan-2 was associated with reduced melanin synthesis, whereas overexpression of syndecan-2 increased melanin synthesis. Similar effects were also detected in human primary epidermal melanocytes. Syndecan-2 expression did not affect the expression of tyrosinase, a key enzyme in melanin synthesis, but instead enhanced the enzymatic activity of tyrosinase by increasing the membrane and melanosome localization of its regulator, protein kinase CβII. Furthermore, UVB caused increased syndecan-2 expression, and this up-regulation of syndecan-2 was required for UVB-induced melanin synthesis. Taken together, these data suggest that syndecan-2 regulates melanin synthesis and could be a potential therapeutic target for treating melanin-associated diseases.

  11. The Yeast Magmas Ortholog Pam16 Has an Essential Function in Fermentative Growth That Involves Sphingolipid Metabolism

    PubMed Central

    Short, Mary K.; Hallett, Joshua P.; Tar, Krisztina; Dange, Thomas; Schmidt, Marion; Moir, Robyn; Willis, Ian M.; Jubinsky, Paul T.

    2012-01-01

    Magmas is a growth factor responsive gene encoding an essential mitochondrial protein in mammalian cells. Pam16, the Magmas ortholog in Saccharomyces cerevisiae, is a component of the presequence translocase-associated motor. A temperature-sensitive allele (pam16-I61N) was used to query an array of non-essential gene-deletion strains for synthetic genetic interactions. The pam16-I61N mutation at ambient temperature caused synthetic lethal or sick phenotypes with genes involved in lipid metabolism, perixosome synthesis, histone deacetylation and mitochondrial protein import. The gene deletion array was also screened for suppressors of the pam16-I61N growth defect to identify compensatory pathways. Five suppressor genes were identified (SUR4, ISC1, IPT1, SKN1, and FEN1) and all are involved in sphingolipid metabolism. pam16-I61N cells cultured in glucose at non-permissive temperatures resulted in rapid growth inhibition and G1 cell cycle arrest, but cell viability was maintained. Altered mitochondria morphology, reduced peroxisome induction in glycerol/ethanol and oleate, and changes in the levels of several sphingolipids including C18 alpha-hydroxy-phytoceramide, were also observed in the temperature sensitive strain. Deletion of SUR4, the strongest suppressor, reversed the temperature sensitive fermentative growth defect, the morphological changes and the elevated levels of C18 alpha-hydroxy phytoceramide in pam16-I61N. Deletion of the other four suppressor genes had similar effects on C18 alpha-hydroxy-phytoceramide levels and restored proliferation to the pam16-I61N strain. In addition, pam16-I61N inhibited respiratory growth, likely by reducing cardiolipin, which is essential for mitochondrial function. Our results suggest that the pleiotropic effects caused by impaired Pam16/Magmas function are mediated in part by changes in lipid metabolism. PMID:22808036

  12. The yeast magmas ortholog pam16 has an essential function in fermentative growth that involves sphingolipid metabolism.

    PubMed

    Short, Mary K; Hallett, Joshua P; Tar, Krisztina; Dange, Thomas; Schmidt, Marion; Moir, Robyn; Willis, Ian M; Jubinsky, Paul T

    2012-01-01

    Magmas is a growth factor responsive gene encoding an essential mitochondrial protein in mammalian cells. Pam16, the Magmas ortholog in Saccharomyces cerevisiae, is a component of the presequence translocase-associated motor. A temperature-sensitive allele (pam16-I61N) was used to query an array of non-essential gene-deletion strains for synthetic genetic interactions. The pam16-I61N mutation at ambient temperature caused synthetic lethal or sick phenotypes with genes involved in lipid metabolism, perixosome synthesis, histone deacetylation and mitochondrial protein import. The gene deletion array was also screened for suppressors of the pam16-I61N growth defect to identify compensatory pathways. Five suppressor genes were identified (SUR4, ISC1, IPT1, SKN1, and FEN1) and all are involved in sphingolipid metabolism. pam16-I61N cells cultured in glucose at non-permissive temperatures resulted in rapid growth inhibition and G1 cell cycle arrest, but cell viability was maintained. Altered mitochondria morphology, reduced peroxisome induction in glycerol/ethanol and oleate, and changes in the levels of several sphingolipids including C18 alpha-hydroxy-phytoceramide, were also observed in the temperature sensitive strain. Deletion of SUR4, the strongest suppressor, reversed the temperature sensitive fermentative growth defect, the morphological changes and the elevated levels of C18 alpha-hydroxy phytoceramide in pam16-I61N. Deletion of the other four suppressor genes had similar effects on C18 alpha-hydroxy-phytoceramide levels and restored proliferation to the pam16-I61N strain. In addition, pam16-I61N inhibited respiratory growth, likely by reducing cardiolipin, which is essential for mitochondrial function. Our results suggest that the pleiotropic effects caused by impaired Pam16/Magmas function are mediated in part by changes in lipid metabolism.

  13. A plasma-membrane E-MAP reveals links of the eisosome with sphingolipid metabolism and endosomal trafficking.

    PubMed

    Aguilar, Pablo S; Fröhlich, Florian; Rehman, Michael; Shales, Mike; Ulitsky, Igor; Olivera-Couto, Agustina; Braberg, Hannes; Shamir, Ron; Walter, Peter; Mann, Matthias; Ejsing, Christer S; Krogan, Nevan J; Walther, Tobias C

    2010-07-01

    The plasma membrane delimits the cell and controls material and information exchange between itself and the environment. How different plasma-membrane processes are coordinated and how the relative abundance of plasma-membrane lipids and proteins is homeostatically maintained are not yet understood. Here, we used a quantitative genetic interaction map, or E-MAP, to functionally interrogate a set of approximately 400 genes involved in various aspects of plasma-membrane biology, including endocytosis, signaling, lipid metabolism and eisosome function. From this E-MAP, we derived a set of 57,799 individual interactions between genes functioning in these various processes. Using triplet genetic motif analysis, we identified a new component of the eisosome, Eis1, and linked the poorly characterized gene EMP70 to endocytic and eisosome function. Finally, we implicated Rom2, a GDP/GTP exchange factor for Rho1 and Rho2, in the regulation of sphingolipid metabolism.

  14. Developmental Regulation across the Life Span: Toward a New Synthesis

    ERIC Educational Resources Information Center

    Haase, Claudia M.; Heckhausen, Jutta; Wrosch, Carsten

    2013-01-01

    How can individuals regulate their own development to live happy, healthy, and productive lives? Major theories of developmental regulation across the life span have been proposed (e.g., dual-process model of assimilation and accommodation; motivational theory of life-span development; model of selection, optimization, and compensation), but they…

  15. Regulation of phospholipid synthesis in phosphatidylserine synthase-deficient (chol) mutants of Saccharomyces cerevisiae

    SciTech Connect

    Letts, V.A.; Henry, S.A.

    1985-08-01

    Saccharomyces cerevisiae mutants, chol, are deficient in the synthesis of the phospholipid phosphatidylserine owing to lowered activity of the membrane-associated enzyme phosphatidylserine synthase. These mutants are auxotrophic for ethanolamine or choline and, in the absence of these supplements, cannot synthesize phosphatidylethanolamine or phosphatidylcholine (PC). The authors exploited these characteristics of the chol mutants to examine the regulation of phospholipid metabolism in S. cerevisiae. Macromolecular synthesis and phospholipid metabolism were examined in chol cells starved for ethanolamine. Coupled to the decline in PC biosynthesis was a simultaneous decrease in the overall rate of phospholipid synthesis. In particular, the rate of synthesis of phosphatidylinositol decreased in parallel with the decline in PC biosynthesis. However, under conditions of ethanolamine deprivation in chol cells, the cytoplasmic enzyme inositol-1-phosphate synthase could not be repressed by exogenous inositol, and the endogenous synthesis of the phospholipid precursor inositol appeared to be elevated. The implications of these findings with respect to the coordinated regulation of phospholipid synthesis are discussed.

  16. Regulation of muscle protein synthesis and the effects of catabolic states.

    PubMed

    Gordon, Bradley S; Kelleher, Andrew R; Kimball, Scot R

    2013-10-01

    Protein synthesis and degradation are dynamically regulated processes that act in concert to control the accretion or loss of muscle mass. The present article focuses on the mechanisms involved in the impairment of protein synthesis that are associated with skeletal muscle atrophy. The vast majority of mechanisms known to regulate protein synthesis involve modulation of the initiation phase of mRNA translation, which comprises a series of reactions that result in the binding of initiator methionyl-tRNAi and mRNA to the 40S ribosomal subunit. The function of the proteins involved in both events has been shown to be repressed under atrophic conditions such as sepsis, cachexia, chronic kidney disease, sarcopenia, and disuse atrophy. The basis for the inhibition of protein synthesis under such conditions is likely to be multifactorial and includes insulin/insulin-like growth factor 1 resistance, pro-inflammatory cytokine expression, malnutrition, corticosteroids, and/or physical inactivity. The present article provides an overview of the existing literature regarding mechanisms and signaling pathways involved in the regulation of mRNA translation as they apply to skeletal muscle wasting, as well as the efficacy of potential clinical interventions such as nutrition and exercise in the maintenance of skeletal muscle protein synthesis under atrophic conditions. This article is part of a Directed Issue entitled: Molecular basis of muscle wasting.

  17. AMPD2 Regulates GTP Synthesis and is Mutated in a Potentially-Treatable Neurodegenerative Brainstem Disorder

    PubMed Central

    Akizu, Naiara; Cantagrel, Vincent; Schroth, Jana; Cai, Na; Vaux, Keith; McCloskey, Douglas; Naviaux, Robert K.; Vleet, Jeremy Van; Fenstermaker, Ali G.; Silhavy, Jennifer L.; Scheliga, Judith S.; Toyama, Keiko; Morisaki, Hiroko; Sonmez, Fatma Mujgan; Celep, Figen; Oraby, Azza; Zaki, Maha S.; Al-Baradie, Raidah; Faqeih, Eissa; Saleh, Mohammad; Spencer, Emily; Rosti, Rasim Ozgur; Scott, Eric; Nickerson, Elizabeth; Gabriel, Stacey; Morisaki, Takayuki; Holmes, Edward W.; Gleeson, Joseph G.

    2013-01-01

    Purine biosynthesis and metabolism, conserved in all living organisms, is essential for cellular energy homeostasis and nucleic acids synthesis. The de novo synthesis of purine precursors is under tight negative feedback regulation mediated by adenosine and guanine nucleotides. We describe a new distinct early-onset neurodegenerative condition resulting from mutations in the adenosine monophosphate deaminase 2 gene (AMPD2). Patients have characteristic brain imaging features of pontocerebellar hypoplasia (PCH), due to loss of brainstem and cerebellar parenchyma. We found that AMPD2 plays an evolutionary conserved role in the maintenance of cellular guanine nucleotide pools by regulating the feedback inhibition of adenosine derivatives on de novo purine synthesis. AMPD2 deficiency results in defective GTP-dependent initiation of protein translation, which can be rescued by administration of purine precursors. These data suggest AMPD2-related PCH as a new, potentially treatable early-onset neurodegenerative disease. PMID:23911318

  18. In vivo regulation of muscle glycogen synthase and the control of glycogen synthesis.

    PubMed Central

    Shulman, R G; Bloch, G; Rothman, D L

    1995-01-01

    The activity of glycogen synthase (GSase; EC 2.4.1.11) is regulated by covalent phosphorylation. Because of this regulation, GSase has generally been considered to control the rate of glycogen synthesis. This hypothesis is examined in light of recent in vivo NMR experiments on rat and human muscle and is found to be quantitatively inconsistent with the data under conditions of glycogen synthesis. Our first experiments showed that muscle glycogen synthesis was slower in non-insulin-dependent diabetics compared to normals and that their defect was in the glucose transporter/hexokinase (GT/HK) part of the pathway. From these and other in vivo NMR results a quantitative model is proposed in which the GT/HK steps control the rate of glycogen synthesis in normal humans and rat muscle. The flux through GSase is regulated to match the proximal steps by "feed forward" to glucose 6-phosphate, which is a positive allosteric effector of all forms of GSase. Recent in vivo NMR experiments specifically designed to test the model are analyzed by metabolic control theory and it is shown quantitatively that the GT/HK step controls the rate of glycogen synthesis. Preliminary evidence favors the transporter step. Several conclusions are significant: (i) glucose transport/hexokinase controls the glycogen synthesis flux; (ii) the role of covalent phosphorylation of GSase is to adapt the activity of the enzyme to the flux and to control the metabolite levels not the flux; (iii) the quantitative data needed for inferring and testing the present model of flux control depended upon advances of in vivo NMR methods that accurately measured the concentration of glucose 6-phosphate and the rate of glycogen synthesis. Images Fig. 1 PMID:7567971

  19. In low protein diets, microRNA-19b regulates urea synthesis by targeting SIRT5

    PubMed Central

    Sun, Rui-Ping; Xi, Qian-Yun; Sun, Jia-Jie; Cheng, Xiao; Zhu, Yan-Ling; Ye, Ding-Ze; Chen, Ting; Wei, Li-Min; Ye, Rui-Song; Jiang, Qing-Yan; Zhang, Yong-Liang

    2016-01-01

    Ammonia detoxification, which takes place via the hepatic urea cycle, is essential for nitrogen homeostasis and physiological well-being. It has been reported that a reduction in dietary protein reduces urea nitrogen. MicroRNAs (miRNAs) are major regulatory non-coding RNAs that have significant effects on several metabolic pathways; however, little is known on whether miRNAs regulate hepatic urea synthesis. The objective of this study was to assess the miRNA expression profile in a low protein diet and identify miRNAs involved in the regulation of the hepatic urea cycle using a porcine model. Weaned 28-days old piglets were fed a corn-soybean normal protein diet (NP) or a corn-soybean low protein diet (LP) for 30 d. Hepatic and blood samples were collected, and the miRNA expression profile was assessed by sequencing and qRT-PCR. Furthermore, we evaluated the possible role of miR-19b in urea synthesis regulation. There were 25 differentially expressed miRNAs between the NP and LP groups. Six of these miRNAs were predicted to be involved in urea cycle metabolism. MiR-19b negatively regulated urea synthesis by targeting SIRT5, which is a positive regulator of CPS1, the rate limiting enzyme in the urea cycle. Our study presented a novel explanation of ureagenesis regulation by miRNAs. PMID:27686746

  20. Newly identified essential amino acid residues affecting ^8-sphingolipid desaturase activity revealed by site-directed mutagenesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In order to identify amino acid residues crucial for the enzymatic activity of ^8-sphingolipid desaturases, a sequence comparison was performed among ^8-sphingolipid desaturases and ^6-fatty acid desaturase from various plants. In addition to the known conserved cytb5 (cytochrome b5) HPGG motif and...

  1. The chemistry and biology of 6-hydroxyceramide, the youngest member of the human sphingolipid family.

    PubMed

    Kováčik, Andrej; Roh, Jaroslav; Vávrová, Kateřina

    2014-07-21

    Sphingolipids are crucial for the life of the cell. In land-dwelling mammals, they are equally important outside the cell-in the extracellular space of the skin barrier-because they prevent loss of water. Although a large body of research has elucidated many of the functions of sphingolipids, their extensive structural diversity remains intriguing. A new class of sphingolipids based on 6-hydroxylated sphingosine has recently been identified in human skin. Abnormal levels of these 6-hydroxylated ceramides have repeatedly been observed in atopic dermatitis; however, neither the biosynthesis nor the roles of these unique ceramide subclasses have been established in the human body. In this Minireview, we summarize the current knowledge of 6-hydroxyceramides, including their discovery, structure, stereochemistry, occurrence in healthy and diseased human epidermis, and synthetic approaches to 6-hydroxysphingosine and related ceramides.

  2. Coordinated regulation of melatonin synthesis and degradation genes in rice leaves in response to cadmium treatment.

    PubMed

    Byeon, Yeong; Lee, Hyoung Yool; Hwang, Ok Jin; Lee, Hye-Jung; Lee, Kyungjin; Back, Kyoungwhan

    2015-05-01

    We investigated the expression patterns of genes involved in melatonin synthesis and degradation in rice leaves upon cadmium (Cd) treatment and the subcellular localization sites of melatonin 2-hydroxylase (M2H) proteins. The Cd-induced synthesis of melatonin coincided with the increased expression of melatonin biosynthetic genes including tryptophan decarboxylase (TDC), tryptamine 5-hydroxylase (T5H), and N-acetylserotonin methyltransferase (ASMT). However, the expression of serotonin N-acetyltransferase (SNAT), the penultimate gene in melatonin biosynthesis, was downregulated, suggesting that melatonin synthesis was counter-regulated by SNAT. Notably, the induction of melatonin biosynthetic gene expression was coupled with the induction of four M2H genes involved in melatonin degradation, which suggests that genes for melatonin synthesis and degradation are coordinately regulated. The induced M2H gene expression was correlated with enhanced M2H enzyme activity. Three of the M2H proteins were localized to the cytoplasm and one M2H protein was localized to chloroplasts, indicating that melatonin degradation occurs both in the cytoplasm and in chloroplasts. The biological activity of 2-hydroxymelatonin in the induction of the plant defense gene expression was 50% less than that of melatonin, which indicates that 2-hydroxymelatonin may be a metabolite of melatonin. Overall, our data demonstrate that melatonin synthesis occurs in parallel with melatonin degradation in both chloroplasts and cytoplasm, and the resulting melatonin metabolite 2-hydroxymelatonin also acts as a signaling molecule for defense gene induction.

  3. Os2 MAP kinase-mediated osmostress tolerance in Penicillium digitatum is associated with its positive regulation on glycerol synthesis and negative regulation on ergosterol synthesis.

    PubMed

    Wang, Mingshuang; Chen, Changsheng; Zhu, Congyi; Sun, Xuepeng; Ruan, Ruoxin; Li, Hongye

    2014-01-01

    High osmolarity glycerol (HOG) pathway is ubiquitously distributed among eukaryotic organisms and plays an important role in adaptation to changes in the environment. In this study, the Hog1 ortholog in Penicillium digitatum, designated Pdos2, was identified and characterized using a gene knock-out strategy. The ΔPdos2 mutant showed a considerably increased sensitivity to salt stress and cell wall-disturbing agents and a slightly increased resistance to fungicides iprodione and fludioxonil, indicating that Pdos2 is involved in response to hyperosmotic stress, regulation of cell wall integrity and sensitivity to fungicides iprodione and fludioxonil. Surprisingly, the mutant was not affected in response to oxidative stress caused by H2O2. The average lesion size in citrus fruits caused by ΔPdos2 mutant was smaller (approximately 25.0% reduction) than that caused by the wild-type strain of P. digitatum at 4 days post inoculation, which suggests that Pdos2 is needed for full virulence of P. digitatum. Interestingly, in the presence of 0.7 M NaCl, the glycerol content was remarkably increased and the ergosterol was decreased in mycelia of the wide-type P. digitatum, whereas the glycerol content was only slightly increased and the ergosterol content remained stable in the ΔPdos2 mutant, suggesting that Pdos2-mediated osmotic adaption is associated with its positive regulation on glycerol synthesis and negative regulation on ergosterol synthesis.

  4. Regulation of dopamine synthesis and release in striatal and prefrontal cortical brain slices

    SciTech Connect

    Wolf, M.E.

    1986-01-01

    Brain slices were used to investigate the role of nerve terminal autoreceptors in modulating dopamine (DA) synthesis and release in striatum and prefrontal cortex. Accumulation of dihydroxyphenylalanine (DOPA) was used as an index of tyrosine hydroxylation in vitro. Nomifensine, a DA uptake blocker, inhibited DOPA synthesis in striatal but not prefrontal slices. This effect was reversed by the DA antagonist sulpiride, suggesting it involved activation of DA receptors by elevated synaptic levels of DA. The autoreceptor-selective agonist EMD-23-448 also inhibited striatal but not prefrontal DOPA synthesis. DOPA synthesis was stimulated in both brain regions by elevated K/sup +/, however only striatal synthesis could be further enhanced by sulpiride. DA release was measured by following the efflux of radioactivity from brain slices prelabeled with (/sup 3/H)-DA. EMD-23-448 and apomorphine inhibited, while sulpiride enhanced, the K/sup +/-evoked overflow of radioactivity from both striatal and prefrontal cortical slices. These findings suggest that striatal DA nerve terminals possess autoreceptors which modulate tyrosine hydroxylation as well as autoreceptors which modulate release. Alternatively, one site may be coupled to both functions through distinct transduction mechanisms. In contrast, autoreceptors on prefrontal cortical terminals appear to regulate DA release but not DA synthesis.

  5. Regulation of sucrose synthesis in water stressed leaves

    SciTech Connect

    Daie, J.; Aloni, B. )

    1991-05-01

    Alteration in carbon metabolism and carbohydrate partitioning occur in drought stressed plants. Some species accumulate large quantities of starch in the chloroplast, which may be used to support sucrose synthesis under conditions of limited carbon supply. The authors monitored chemical partitioning of carbon between sugars and starch and the activity of sucrose phosphate synthase (SPS) and fructose 1,6 bisphosphatase (FBPase) in the source leaves of water stressed tomatoes. Plants were stressed by withdrawing water for 10 days and rewatered for recovery. Water potential dropped from {minus}0.8 to {minus}2.2MPA in 10 days, but recovered to control level 2 days after rewatering. Photosynthetic rates as measured by the activity of Rubisco followed similar patterns to those of water potential. After 10 days, leaf starch levels decreased to less than 50% of control. Sucrose levels did not increase significantly, but hexose levels increased 3-4 fold during the stress period, and decreased to control levels 1 day after rewatering. FBPase activity decreased and SPS activity increased under stress conditions. Upon rewatering, the activity of FBPase and SPS returned to control levels. Presence of large quantities of hexose and activation of SPS in stressed leaves suggested that additional sucrose synthesized under stress was hydrolyzed to hexoses, presumably due to enhanced invertase activity.

  6. Regulation of phosphatidylcholine synthesis in rat liver endoplasmic reticulum.

    PubMed Central

    Sribney, M; Knowles, C L; Lyman, E M

    1976-01-01

    The biosynthesis of phosphatidylcholine in rat liver microsomal preparations catalysed by CDP-choline-1,2-diacylglycerol cholinephosphotransferase (EC 2.7.8.2) was inhibited by a combination of ATP and CoA or ATP and pantetheine. ATP alone at high concentrations (20 mM) inhibits phosphatidylcholine formation to the extent of 70%. In the presence of 0.1 mM-CoA, ATP (2 mM) inhibits to the extent of 80% and in the presence of 1 mM-pantetheine to the extent of 90%. ADP and other nucleotide triphosphates in combination with either CoA or pantetheine are only 10-30% as effective in inhibiting phosphatidylcholine synthesis. AMP(CH2)PP [adenosine 5'-(alphabeta-methylene)triphosphate] together with CoA inhibits to the extent of 59% and with pantetheine by 48%. AMP-P(CH2)P [adenosine 5'-(betagamma-methylene)triphosphate] together with either CoA or pantetheine had no significant effect on phosphatidylcholine formation. Other closely related derivatives of pantothenic acid were without effect either alone or in the presence of ATP, as were thiol compounds such as cysteine, homocysteine, cysteamine, dithiothreitol and glutathione. Several mechanisms by which this inhibition might take place were ruled out and it is concluded that ATP together with either CoA or pantetheine interacts reversibly with phosphatidylcholine synthetase to cause temporarily the inhibition of phosphatidylcholine formation. PMID:182154

  7. Regulation of the salvage pathway of deoxynucleotides synthesis in apoptosis induced by growth factor deprivation.

    PubMed Central

    Oliver, F J; Collins, M K; López-Rivas, A

    1996-01-01

    Here we describe changes in dNTP metabolism that precede DNA fragmentation in a model of apoptosis driven by deprivation of the cytokine interleukin 3 (IL-3). In haemopoietic BAF3 cells, IL-3 withdrawal leads to a rapid decrease in the size of dATP, dTTP and dGTP pools without affecting dCTP levels. This imbalance in dNTP pools precedes DNA fragmentation and is accompanied by down-regulation of enzymes controlling the de novo and salvage pathways of dNTP synthesis, ribonucleotide reductase and thymidine kinase (TK) respectively. Readdition of IL-3 results in a rapid, protein synthesis-independent restoration of normal dNTP pools, enhanced TK activity and increased precursor incorporation through the salvage pathway. Up-regulation of TK activity after IL-3 readdition is prevented by the protein kinase C (PKC) inhibitor staurosporin, but not by tyrosine kinase inhibitors. Furthermore activation of PKC by phorbol esters mimics the stimulatory effect of IL-3 on TK activity, suggesting that PKC might be involved in regulating this effect. These results indicate that regulation by IL-3 of the salvage pathway of dNTP synthesis plays a role in the maintenance of cellular dNTP pool balance and suggests that alterations in dNTP metabolism after IL-3 deprivation could be a relevant event in the commitment of haemopoietic cells to apoptosis. PMID:8687383

  8. MiR-19a regulates PTEN expression to mediate glycogen synthesis in hepatocytes

    PubMed Central

    Dou, Lin; Meng, Xiangyu; Sui, Xiaofang; Wang, Shuyue; Shen, Tao; Huang, Xiuqing; Guo, Jun; Fang, Weiwei; Man, Yong; Xi, Jianzhong; Li, Jian

    2015-01-01

    MiR-19a, a member of mir-17-92 microRNA clusters, has been demonstrated to promote cell proliferation and angiogenesis via regulating the PI3K/AKT pathway, the major insulin signaling pathway. However, whether miR-19a plays an important role in glycogen synthesis in hepatocytes remains unknown. Here, we define the impact of miR-19a on glycogen synthesis and IL-6-induced reduced glycogenesis in hepatocytes and its underlying mechanisms. Our studies indicate that miR-19a was down-regulated in the livers of db/db mice and mice injected with IL-6, as well as mouse NCTC 1469 hepatocytes and HEP 1–6 hepatocytes treated by IL-6. We found that over-expression of miR-19a in NCTC 1469 cells and HEP 1–6 cells led to increased activation of the AKT/GSK pathway and synthesis of glycogen, whereas down-regulation of miR-19a impaired AKT/GSK phosphorylation and glycogenesis. Over-expression of miR-19a ameliorated IL-6-induced reduced glycogen synthesis in hepatocytes. Moreover, we identified PTEN as the target of miR-19a by a luciferase assay. Down-regulation of PTEN rescued the effects of miR-19a suppression on the activation of the AKT/GSK pathway and improved glycogenesis in NTC 1469 cells. These findings show for the first time that miR-19a might activate the AKT/GSK pathway and glycogenesis via down-regulation of PTEN expression. PMID:26111969

  9. Extra-adrenal glucocorticoid synthesis: immune regulation and aspects on local organ homeostasis.

    PubMed

    Talabér, Gergely; Jondal, Mikael; Okret, Sam

    2013-11-05

    Systemic glucocorticoids (GCs) mainly originate from de novo synthesis in the adrenal cortex under the control of the hypothalamus-pituitary-adrenal (HPA)-axis. However, research during the last 1-2 decades has revealed that additional organs express the necessary enzymes and have the capacity for de novo synthesis of biologically active GCs. This includes the thymus, intestine, skin and the brain. Recent research has also revealed that locally synthesized GCs most likely act in a paracrine or autocrine manner and have significant physiological roles in local homeostasis, cell development and immune cell activation. In this review, we summarize the nature, regulation and known physiological roles of extra-adrenal GC synthesis. We specifically focus on the thymus in which GC production (by both developing thymocytes and epithelial cells) has a role in the maintenance of proper immunological function.

  10. PERK Regulates Working Memory and Protein Synthesis-Dependent Memory Flexibility.

    PubMed

    Zhu, Siying; Henninger, Keely; McGrath, Barbara C; Cavener, Douglas R

    2016-01-01

    PERK (EIF2AK3) is an ER-resident eIF2α kinase required for memory flexibility and metabotropic glutamate receptor-dependent long-term depression, processes known to be dependent on new protein synthesis. Here we investigated PERK's role in working memory, a cognitive ability that is independent of new protein synthesis, but instead is dependent on cellular Ca2+ dynamics. We found that working memory is impaired in forebrain-specific Perk knockout and pharmacologically PERK-inhibited mice. Moreover, inhibition of PERK in wild-type mice mimics the fear extinction impairment observed in forebrain-specific Perk knockout mice. Our findings reveal a novel role of PERK in cognitive functions and suggest that PERK regulates both Ca2+ -dependent working memory and protein synthesis-dependent memory flexibility.

  11. Impact of insulin deprivation and treatment on sphingolipid distribution in different muscle subcellular compartments of streptozotocin-diabetic C57Bl/6 mice

    PubMed Central

    Zabielski, Piotr; Blachnio-Zabielska, Agnieszka; Lanza, Ian R.; Gopala, Srinivas; Manjunatha, S.; Jakaitis, Daniel R.; Persson, Xuan-Mai; Gransee, Jaime; Klaus, Katherine A.; Schimke, Jill M.; Jensen, Michael D.

    2013-01-01

    Insulin deprivation in type 1 diabetes (T1D) individuals increases lipolysis and plasma free fatty acids (FFA) concentration, which can stimulate synthesis of intramyocellular bioactive lipids such as ceramides (Cer) and long-chain fatty acid-CoAs (LCFa-CoAs). Ceramide was shown to decrease muscle insulin sensitivity, and at mitochondrial levels it stimulates reactive oxygen species production. Here, we show that insulin deprivation in streptozotocin diabetic C57BL/6 mice increases quadriceps muscle Cer content, which was correlated with a concomitant decrease in the body fat and increased plasma FFA, glycosylated hemoglobin level (%Hb A1c), and muscular LCFa-CoA content. The alternations were accompanied by an increase in protein expression in LCFa-CoA and Cer synthesis (FATP1/ACSVL5, CerS1, CerS5), a decrease in the expression of genes implicated in muscle insulin sensitivity (GLUT4, GYS1), and inhibition of insulin signaling cascade by Aktα and GYS3β phosphorylation under acute insulin stimulation. Both the content and composition of sarcoplasmic fraction sphingolipids were most affected by insulin deprivation, whereas mitochondrial fraction sphingolipids remained stable. The observed effects of insulin deprivation were reversed, except for content and composition of LCFa-CoA, CerS protein expression, GYS1 gene expression, and phosphorylation status of Akt and GYS3β when exogenous insulin was provided by subcutaneous insulin implants. Principal component analysis and Pearson's correlation analysis revealed close relationships between the features of the diabetic phenotype, the content of LCFa-CoAs and Cers containing C18-fatty acids in sarcoplasm, but not in mitochondria. Insulin replacement did not completely rescue the phenotype, especially regarding the content of LCFa-CoA, or proteins implicated in Cer synthesis and muscle insulin sensitivity. These persistent changes might contribute to muscle insulin resistance observed in T1D individuals. PMID:24368672

  12. Regulation of IL-13 synthesis in human lymphocytes: implications for asthma therapy

    PubMed Central

    Pahl, Andreas; Zhang, Meixia; Kuss, Hildegard; Szelenyi, Istvan; Brune, Kay

    2002-01-01

    IL-13 is an important mediator in inflammatory diseases such as asthma. IL-13 is mainly produced by T cells. However, signalling pathways leading to induction of this cytokine are not well-characterized. We analysed the regulation of IL-13 in human peripheral blood mononuclear cells and CD4+ T cells.Cyclosporine (CsA) and FK-506 inhibited IL-13 synthesis, when cells were stimulated by TPA/ionomycin. However, stimulation by α-CD3/α-CD28 led to an enhanced IL-13 synthesis.NF-κB inhibitor N-tosyl-L-lysine chloromethylketone (TLCK) inhibited IL-13 synthesis more effectively after TPA/ionomycin stimulation. After α-CD3/α-CD28 stimulation, only 300 μM TLCK inhibited IL-13 synthesis. Dexamethasone inhibited IL-13 equally effective after α-CD3/α-CD28 and TPA/ionomycin stimulation.p38 MAPK inhibitor SB203580 inhibited IL-13 synthesis only partially. MEK inhibitor U0126 inhibited TPA/ionomycin induced IL-13 synthesis very effectively, whereas α-CD3/α-CD28 stimulated IL-13 induction was resistant to this drug.These results were confirmed in purified CD4+ T cells. In difference to PBMCs α-CD3/α-CD28 stimulated IL-13 synthesis was effectively inhibited by CsA, FK-506 and U0126.Therefore U0126 was tested in an animal model of allergic asthma. We could demonstrate for the first time that inhibition of the MEK – ERK cascade is a therapeutic option for asthma. Intraperitoneal administration of 10 mg kg−1 U0126 reduced lung eosinophilia in ovalbumin-challenged Brown Norway rats by 44%.These results demonstrate that different signalling pathways are involved in regulating IL-13 synthesis in primary human T cells. Characterizing highly potent inhibitors of IL-13 synthesis can be exploited to identify new drugs to treat immunological diseases such as asthma. PMID:11959794

  13. Genetic, biochemical, and transcriptional responses of Saccharomyces cerevisiae to the novel immunomodulator FTY720 largely mimic those of the natural sphingolipid phytosphingosine.

    PubMed

    Welsch, Carole A; Roth, Lukas W A; Goetschy, Jean François; Movva, N Rao

    2004-08-27

    Sphingolipids are signaling molecules that influence diverse cellular functions from control of the cell cycle to degradation of plasma membrane proteins. The synthetic sphingolipid-like compound FTY720 is an immunomodulating agent in clinical trials for transplant graft maintenance. In this report, we compare the effects of the natural yeast sphingolipid phytosphingosine with FTY720 in Saccharomyces cerevisiae. We show that the multicopy suppressor genes that induce growth resistance to FTY720 also confer resistance to growth-inhibitory concentrations of phytosphingosine. In addition, mutants for ubiquitination pathway proteins are shown to be resistant to the growth-inhibiting effect of both FTY720 and phytosphingosine. We observe fewer similarities between sphingosine and FTY720 than between FTY720 and phytosphingosine as revealed by genetic studies. Yeast cells lacking the specific sphingosine kinase LCB4 are sensitive to phytosphingosine and FTY720 but resistant to sphingosine, suggesting that FTY720 and phytosphingosine have a more related mechanism of action. Gene expression profile comparisons of sensitive and resistant yeast cells exposed to FTY720 and phytosphingosine highlight a number of similarities. In response to treatment with these compounds, approximately 77% of the genes that are regulated >2-fold by FTY720 also respond to phytosphingosine in the same direction in the parent strain. In addition, a close inspection of TAT1 and TAT2 transporters following exposure to phytosphingosine indicates that TAT1 protein is degraded in a similar fashion upon treatment with FTY720 and phytosphingosine. There were differences, however, with respect to the TAT2 protein level and the expression profiles of a subset of genes. The genetic, transcriptional, and biochemical data together indicate that FTY720 and phytosphingosine influence similar pathways in yeast cells. These findings offer further insights into the physiological pathways influenced by these

  14. Skin ceramide alterations in first-episode schizophrenia indicate abnormal sphingolipid metabolism.

    PubMed

    Smesny, Stefan; Schmelzer, Christian E H; Hinder, Anke; Köhler, Alexandra; Schneider, Christiane; Rudzok, Maria; Schmidt, Ulrike; Milleit, Berko; Milleit, Christine; Nenadic, Igor; Sauer, Heinrich; Neubert, Reinhard H H; Fluhr, Joachim W

    2013-07-01

    There is considerable evidence for specific pathology of lipid metabolism in schizophrenia, affecting polyunsaturated fatty acids and in particular sphingolipids. These deficits are assumed to interfere with neuronal membrane functioning and the development and maintenance of myelin sheaths. Recent studies suggest that some of these lipid pathologies might also be detected in peripheral skin tests. In this study, we examined different skin lipids and their relation to schizophrenia. We assessed epidermal lipid profiles in 22 first-episode antipsychotic-naïve schizophrenia patients and 22 healthy controls matched for age and gender using a hexan/ethanol extraction technique and combined high-performance thin-layer chromatography/gas-chromatography. We found highly significant increase of ceramide AH and NH/AS classes in patients and decrease of EOS and NP ceramide classes. This is the first demonstration of specific peripheral sphingolipid alterations in schizophrenia. The results support recent models of systemic lipid pathology and in particular of specific sphingolipids, which are crucial in neuronal membrane integrity. Given recent findings showing amelioration of psychopathology using fatty acid supplementation, our findings also bear relevance for sphingolipids as potential biomarkers of the disease.

  15. Toxoplasma gondii salvages sphingolipids from the host Golgi through the rerouting of selected Rab vesicles to the parasitophorous vacuole

    PubMed Central

    Romano, Julia D.; Sonda, Sabrina; Bergbower, Emily; Smith, Maria Elisa; Coppens, Isabelle

    2013-01-01

    The obligate intracellular protozoan Toxoplasma gondii actively invades mammalian cells and, upon entry, forms its own membrane-bound compartment, named the parasitophorous vacuole (PV). Within the PV, the parasite replicates and scavenges nutrients, including lipids, from host organelles. Although T. gondii can synthesize sphingolipids de novo, it also scavenges these lipids from the host Golgi. How the parasite obtains sphingolipids from the Golgi remains unclear, as the PV avoids fusion with host organelles. In this study, we explore the host Golgi–PV interaction and evaluate the importance of host-derived sphingolipids for parasite growth. We demonstrate that the PV preferentially localizes near the host Golgi early during infection and remains closely associated with this organelle throughout infection. The parasite subverts the structure of the host Golgi, resulting in its fragmentation into numerous ministacks, which surround the PV, and hijacks host Golgi–derived vesicles within the PV. These vesicles, marked with Rab14, Rab30, or Rab43, colocalize with host-derived sphingolipids in the vacuolar space. Scavenged sphingolipids contribute to parasite replication since alterations in host sphingolipid metabolism are detrimental for the parasite's growth. Thus our results reveal that T. gondii relies on host-derived sphingolipids for its development and scavenges these lipids via Golgi-derived vesicles. PMID:23615442

  16. Toxoplasma gondii salvages sphingolipids from the host Golgi through the rerouting of selected Rab vesicles to the parasitophorous vacuole.

    PubMed

    Romano, Julia D; Sonda, Sabrina; Bergbower, Emily; Smith, Maria Elisa; Coppens, Isabelle

    2013-06-01

    The obligate intracellular protozoan Toxoplasma gondii actively invades mammalian cells and, upon entry, forms its own membrane-bound compartment, named the parasitophorous vacuole (PV). Within the PV, the parasite replicates and scavenges nutrients, including lipids, from host organelles. Although T. gondii can synthesize sphingolipids de novo, it also scavenges these lipids from the host Golgi. How the parasite obtains sphingolipids from the Golgi remains unclear, as the PV avoids fusion with host organelles. In this study, we explore the host Golgi-PV interaction and evaluate the importance of host-derived sphingolipids for parasite growth. We demonstrate that the PV preferentially localizes near the host Golgi early during infection and remains closely associated with this organelle throughout infection. The parasite subverts the structure of the host Golgi, resulting in its fragmentation into numerous ministacks, which surround the PV, and hijacks host Golgi-derived vesicles within the PV. These vesicles, marked with Rab14, Rab30, or Rab43, colocalize with host-derived sphingolipids in the vacuolar space. Scavenged sphingolipids contribute to parasite replication since alterations in host sphingolipid metabolism are detrimental for the parasite's growth. Thus our results reveal that T. gondii relies on host-derived sphingolipids for its development and scavenges these lipids via Golgi-derived vesicles.

  17. Mechanisms regulating melatonin synthesis in the mammalian pineal organ.

    PubMed

    Schomerus, Christof; Korf, Horst-Werner

    2005-12-01

    The day/night rhythm in melatonin production is a characteristic feature in vertebrate physiology. This hormonal signal reliably reflects the environmental light conditions and is independent of behavioral aspects. In all mammalian species, melatonin production is regulated by norepinephrine, which is released from sympathetic nerve fibers exclusively at night. Norepinephrine elevates the intracellular cAMP concentration via beta-adrenergic receptors and activates the cAMP-dependent protein kinase A. This pathway is crucial for regulation of the penultimate enzyme in melatonin biosynthesis, the arylalkylamine N-acetyltransferase (AANAT); cAMP/protein kinase A may, however, act in different ways. In ungulates and primates, pinealocytes constantly synthesize AANAT protein from continually available Aanat mRNA. During the day-in the absence of noradrenergic stimulation-the protein is immediately destroyed by proteasomal proteolysis. At nighttime, elevated cAMP levels cause phosphorylation of AANAT by protein kinase A. This posttranslational modification leads to interaction of phosphorylated AANAT with regulatory 14-3-3 proteins, which protect AANAT from degradation. Increases in AANAT protein are paralleled by increases in enzyme activity. Stimulation of the cAMP/protein kinase A pathway may also activate pineal gene expression. In rodents, transcriptional activation of the Aanat gene is the primary mechanism for the induction of melatonin biosynthesis and results in marked day/night fluctuations in Aanat mRNA. It involves protein kinase A-dependent phosphorylation of the transcription factor cyclic AMP response element-binding protein (CREB) and binding of phosphorylated CREB in the promoter region of the Aanat gene. In conclusion, a common neuroendocrine principle, the nocturnal rise in melatonin, is controlled by strikingly diverse regulatory mechanisms. This diversity has emerged in the course of evolution and reflects the high adaptive plasticity of the

  18. MicroRNA and AU-rich element regulation of prostaglandin synthesis

    PubMed Central

    Moore, Ashleigh E.; Young, Lisa E.

    2012-01-01

    Many liness of evidence demonstrate that prostaglandins play an important role in cancer, and enhanced synthesis of prostaglandin E2 (PGE2) is often observed in various human malignancies often associated with poor prognosis. PGE2 synthesis is initiated with the release of arachidonic acid by phospholipase enzymes, where it is then converted into the intermediate prostaglandin prostaglandin H2 (PGH2) by members of the cyclooxygenase family. The synthesis of PGE2 from PGH2 is facilitated by three different PGE synthases, and functional PGE2 can promote tumor growth by binding to four EP receptors to activate signaling pathways that control cell proliferation, migration, apoptosis, and angiogenesis. An integral method of controlling gene expression is by posttranscriptional mechanisms that regulate mRNA stability and protein translation. Messenger RNA regulatory elements typically reside within the 3′ untranslated region (3′UTR) of the transcript and play a critical role in targeting specific mRNAs for posttranscriptional regulation through micro-RNA (miRNA) binding and adenylate- and uridylate-rich element RNA-binding proteins. In this review, we highlight the current advances in our understanding of the impact these RNA sequence elements have upon regulating PGE2 levels. We also identify various RNA sequence elements consistently observed within the 3′UTRs of the genes involved in the PGE2 pathway, indicating these binding sites for miRNAs and RNA-binding proteins to be central regulators of PGE2 synthesis and function. These findings may provide a rationale for the development of new therapeutic approaches to control tumor growth and metastasis promoted by elevated PGE2 levels. PMID:22005950

  19. Serum sphingolipids: relationships to insulin sensitivity and changes with exercise in humans.

    PubMed

    Bergman, Bryan C; Brozinick, Joseph T; Strauss, Allison; Bacon, Samantha; Kerege, Anna; Bui, Hai Hoang; Sanders, Phil; Siddall, Parker; Kuo, Ming Shang; Perreault, Leigh

    2015-08-15

    Ceramides and sphingolipids are a family of lipid molecules that circulate in serum and accumulate in skeletal muscle, promoting insulin resistance. Plasma ceramide and dihydroceramide are related to insulin resistance, yet less is known regarding other ceramide and sphingolipid species. Despite its association with insulin sensitivity, chronic endurance exercise training does not change plasma ceramide and sphingolipid content, with little known regarding a single bout of exercise. We measured basal relationships and the effect of acute exercise (1.5 h at 50% V̇o2 max) and recovery on serum ceramide and sphingolipid content in sedentary obese individuals, endurance-trained athletes, and individuals with type 2 diabetes (T2D). Basal serum C18:0, C20:0, and C24:1 ceramide and C18:0 and total dihydroceramide were significantly higher in T2D and, along with C16:0 ceramide and C18:0 sphingomyelin, correlated positively with insulin resistance. Acute exercise significantly increased serum ceramide, glucosylceramide, and GM3 gangliosides, which largely decreased to basal values in recovery. Sphingosine 1-phosphate and sphingomyelin did not change during exercise but decreased below basal values in recovery. Serum C16:0 and C18:0 ceramide and C18:0 sphingomyelin, but not the total concentrations of either of them, were positively correlated with markers of muscle NF-κB activation, suggesting that specific species activate intracellular inflammation. Interestingly, a subset of sphingomyelin species, notably C14:0, C22:3, and C24:4 species, was positively associated with insulin secretion and glucose tolerance. Together, these data show that unique ceramide and sphingolipid species associate with either protective or deleterious features for diabetes and could provide novel therapeutic targets for the future.

  20. An apparent association between glycosylphosphatidylinositol-anchored proteins and a sphingolipid in Tetrahymena mimbres.

    PubMed Central

    Zhang, X; Thompson, G A

    1997-01-01

    Sphingolipids are thought to stabilize glycosylphosphatidylinositol (GPI)-anchored protein-rich membrane domains of yeast and polarized higher animal cells during the processing and targeting of these proteins to the plasma membrane. A widely used criterion for identifying the stable sphingolipid- and GPI-anchored protein-enriched membrane domains is the resistance of these lipid-modified proteins to solubilization by the detergent Triton X-100 (TX-100) at low temperature. Surprisingly, there have been no reports of sphingolipid/GPI-anchored protein association in protozoans, despite the fact that these cells contain considerably higher levels of GPI-anchored proteins than does any other organism. We report here the presence in Tetrahymena mimbres of a significant pool of GPI-anchored proteins which resisted extraction by 1% TX-100 at 4 degrees C but not at 37 degrees C. Of the total cellular complement of GPI-anchored proteins, which together accounted for more than 2% of whole-cell protein and were especially enriched in surface membranes, 10% of the major 63kDa component (gpi63) and 23% of a somewhat less abundant component (gpi23) were insoluble in TX-100 at 4 degrees C. A substantial proportion of the cell's only abundant sphingolipid, ceramideaminoethylphosphonate (CAEP), was also insoluble in 1% TX-100 at 4 degrees C. Radiolabelling studies involving [3H]leucine incorporation into proteins and [3H]palmitic acid incorporation into lipids revealed that the TX-100-resistant gpi63, gpi23 and CAEP molecules were all metabolically distinct from their TX-100-soluble counterparts in other compartments of the cell. The presence of detergent-resistant sphingolipid/GPI-anchored protein domains in non-polarized ciliate and trypanosomatid cells was probably obscured in previous studies by the profusion of accompanying detergent-soluble molecules. PMID:9173882

  1. Keratinocyte-derived Laminin-332 Protein Promotes Melanin Synthesis via Regulation of Tyrosine Uptake*

    PubMed Central

    Chung, Heesung; Jung, Hyejung; Lee, Jung-hyun; Oh, Hye Yun; Kim, Ok Bin; Han, Inn-Oc; Oh, Eok-Soo

    2014-01-01

    Melanocytes, which produce the pigment melanin, are known to be closely regulated by neighboring keratinocytes. However, how keratinocytes regulate melanin production is unclear. Here we report that melanin production in melanoma cells (B16F10 and MNT-1) was increased markedly on a keratinocyte-derived extracellular matrix compared with a melanoma cell-derived extracellular matrix. siRNA-mediated reduction of keratinocyte-derived laminin-332 expression decreased melanin synthesis in melanoma cells, and laminin-332, but not fibronectin, enhanced melanin content and α-melanocyte-stimulating hormone-regulated melanin production in melanoma cells. Similar effects were observed in human melanocytes. Interestingly, however, laminin-332 did not affect the expression or activity of tyrosinase. Instead, laminin-332 promoted the uptake of extracellular tyrosine and, subsequently, increased intracellular levels of tyrosine in both melanocytes and melanoma cells. Taken together, these data strongly suggest that keratinocyte-derived laminin-332 contributes to melanin production by regulating tyrosine uptake. PMID:24951591

  2. Keratinocyte-derived laminin-332 protein promotes melanin synthesis via regulation of tyrosine uptake.

    PubMed

    Chung, Heesung; Jung, Hyejung; Lee, Jung-Hyun; Oh, Hye Yun; Kim, Ok Bin; Han, Inn-Oc; Oh, Eok-Soo

    2014-08-01

    Melanocytes, which produce the pigment melanin, are known to be closely regulated by neighboring keratinocytes. However, how keratinocytes regulate melanin production is unclear. Here we report that melanin production in melanoma cells (B16F10 and MNT-1) was increased markedly on a keratinocyte-derived extracellular matrix compared with a melanoma cell-derived extracellular matrix. siRNA-mediated reduction of keratinocyte-derived laminin-332 expression decreased melanin synthesis in melanoma cells, and laminin-332, but not fibronectin, enhanced melanin content and α-melanocyte-stimulating hormone-regulated melanin production in melanoma cells. Similar effects were observed in human melanocytes. Interestingly, however, laminin-332 did not affect the expression or activity of tyrosinase. Instead, laminin-332 promoted the uptake of extracellular tyrosine and, subsequently, increased intracellular levels of tyrosine in both melanocytes and melanoma cells. Taken together, these data strongly suggest that keratinocyte-derived laminin-332 contributes to melanin production by regulating tyrosine uptake.

  3. Chlamydia trachomatis Intercepts Golgi-Derived Sphingolipids through a Rab14-Mediated Transport Required for Bacterial Development and Replication

    PubMed Central

    Capmany, Anahí; Damiani, María Teresa

    2010-01-01

    Chlamydia trachomatis are obligate intracellular bacteria that survive and replicate in a bacterial-modified phagosome called inclusion. As other intracellular parasites, these bacteria subvert the phagocytic pathway to avoid degradation in phagolysosomes and exploit trafficking pathways to acquire both energy and nutrients essential for their survival. Rabs are host proteins that control intracellular vesicular trafficking. Rab14, a Golgi-related Rab, controls Golgi to endosomes transport. Since Chlamydia establish a close relationship with the Golgi apparatus, the recruitment and participation of Rab14 on inclusion development and bacteria growth were analyzed. Time course analysis revealed that Rab14 associated with inclusions by 10 h post infection and was maintained throughout the entire developmental cycle. The recruitment was bacterial protein synthesis-dependent but independent of microtubules and Golgi integrity. Overexpression of Rab14 dominant negative mutants delayed inclusion enlargement, and impaired bacteria replication as determined by IFU. Silencing of Rab14 by siRNA also decreased bacteria multiplication and infectivity. By electron microscopy, aberrant bacteria were observed in cells overexpressing the cytosolic negative Rab14 mutant. Our results showed that Rab14 facilitates the delivery of sphingolipids required for bacterial development and replication from the Golgi to chlamydial inclusions. Novel anti-chlamydial therapies could be developed based on the knowledge of how bacteria subvert host vesicular transport events through Rabs manipulation. PMID:21124879

  4. Oxygen and pH regulation of protein synthesis in mitochondria from Artemia franciscana embryos.

    PubMed Central

    Kwast, K E; Hand, S C

    1996-01-01

    To identify factors responsible for the down-regulation of mitochondrial biosynthetic processes during anoxia in encysted Artemia franciscana embryos, the effects of oxygen limitation and pH on protein synthesis were investigated in isolated mitochondria. At the optimal pH of 7.5, exposure of mitochondria to anoxia decreases the protein synthesis rate by 79%. Rates were suppressed by a further 10% at pH 6.8, the intracellular pH (pHi) measured under anoxia in vivo. Matrix pH, measured under identical conditions, was 8.43 +/- 0.01 at an extra-mitochondrial pH of 7.9 (mean +/- S.E.M., n = 3), 8.05 +/- 0.01 at pH 7.5, and 7.10 +/- 0.01 at pH 6.8. The matrix pH did not vary (P > or = 0.20) as a function of oxygen availability during the 1 h assays. Intramitochondrial purine nucleotides varied little as a function of pH. In contrast, after 1 h of protein synthesis under anoxia, ATP levels decreased by up to 40%, whereas AMP, ADP and GDP concentrations increased, and GTP and GMP concentrations remained relatively constant. The addition of 1 mM ATP at the onset of anoxia maintained the ATP/ADP ratio at the aerobic value, but did not stabilized the GTP/GDP ratio or rescue rates of protein synthesis. Thus, at present, we cannot eliminate the possibility that the decrease in the GTP/GDP ratio during anoxia may contribute to the suppression of protein synthesis. The effect of anoxia was reversible; the rate of protein synthesis upon reoxygenation after a 30 min bout of anoxia was comparable (P = 0.14) with the pre-anoxic rate (193 +/- 17 and 174 +/- 6 pmol of leucine per mg of protein respectively, mean +/- S.E.M., n = 3). The array of mitochondrial translation products did not differ qualitatively as a function of either oxygen availability or pH. Finally, similar pH profiles for protein synthesis were obtained with either [3H]leucine or [3H]histidine (known to use different transporters). Consequently, it is improbable that the pH-sensitivity of protein synthesis can be

  5. Xylosyltransferase-I regulates glycosaminoglycan synthesis during the pathogenic process of human osteoarthritis.

    PubMed

    Venkatesan, Narayanan; Barré, Lydia; Bourhim, Mustapha; Magdalou, Jacques; Mainard, Didier; Netter, Patrick; Fournel-Gigleux, Sylvie; Ouzzine, Mohamed

    2012-01-01

    Loss of glycosaminoglycan (GAG) chains of proteoglycans (PGs) is an early event of osteoarthritis (OA) resulting in cartilage degradation that has been previously demonstrated in both huma and experimental OA models. However, the mechanism of GAG loss and the role of xylosyltransferase-I (XT-I) that initiates GAG biosynthesis onto PG molecules in the pathogenic process of human OA are unknown. In this study, we have characterized XT-I expression and activity together with GAG synthesis in human OA cartilage obtained from different regions of the same joint, defined as "normal", "late-stage" or adjacent to "late-stage". The results showed that GAG synthesis and content increased in cartilage from areas flanking OA lesions compared to cartilage from macroscopically "normal" unaffected regions, while decreased in "late-stage" OA cartilage lesions. This increase in anabolic state was associated with a marked upregulation of XT-I expression and activity in cartilage "next to lesion" while a decrease in the "late-stage" OA cartilage. Importantly, XT-I inhibition by shRNA or forced-expression with a pCMV-XT-I construct correlated with the modulation of GAG anabolism in human cartilage explants. The observation that XT-I gene expression was down-regulated by IL-1β and up-regulated by TGF-β1 indicates that these cytokines may play a role in regulating GAG content in human OA. Noteworthy, expression of IL-1β receptor (IL-1R1) was down-regulated whereas that of TGF-β1 was up-regulated in early OA cartilage. Theses observations may account for upregulation of XT-I and sustained GAG synthesis prior to the development of cartilage lesions during the pathogenic process of OA.

  6. Xylosyltransferase-I Regulates Glycosaminoglycan Synthesis during the Pathogenic Process of Human Osteoarthritis

    PubMed Central

    Venkatesan, Narayanan; Barré, Lydia; Bourhim, Mustapha; Magdalou, Jacques; Mainard, Didier; Netter, Patrick; Fournel-Gigleux, Sylvie; Ouzzine, Mohamed

    2012-01-01

    Loss of glycosaminoglycan (GAG) chains of proteoglycans (PGs) is an early event of osteoarthritis (OA) resulting in cartilage degradation that has been previously demonstrated in both huma and experimental OA models. However, the mechanism of GAG loss and the role of xylosyltransferase-I (XT-I) that initiates GAG biosynthesis onto PG molecules in the pathogenic process of human OA are unknown. In this study, we have characterized XT-I expression and activity together with GAG synthesis in human OA cartilage obtained from different regions of the same joint, defined as “normal”, “late-stage” or adjacent to “late-stage”. The results showed that GAG synthesis and content increased in cartilage from areas flanking OA lesions compared to cartilage from macroscopically “normal” unaffected regions, while decreased in “late-stage” OA cartilage lesions. This increase in anabolic state was associated with a marked upregulation of XT-I expression and activity in cartilage “next to lesion” while a decrease in the “late-stage” OA cartilage. Importantly, XT-I inhibition by shRNA or forced-expression with a pCMV-XT-I construct correlated with the modulation of GAG anabolism in human cartilage explants. The observation that XT-I gene expression was down-regulated by IL-1β and up-regulated by TGF-β1 indicates that these cytokines may play a role in regulating GAG content in human OA. Noteworthy, expression of IL-1β receptor (IL-1R1) was down-regulated whereas that of TGF-β1 was up-regulated in early OA cartilage. Theses observations may account for upregulation of XT-I and sustained GAG synthesis prior to the development of cartilage lesions during the pathogenic process of OA. PMID:22479506

  7. miRNAs Do Not Regulate Circadian Protein Synthesis in the Dinoflagellate Lingulodinium polyedrum

    PubMed Central

    Dagenais-Bellefeuille, Steve; Beauchemin, Mathieu; Morse, David

    2017-01-01

    Dinoflagellates have been shown to express miRNA by bioinformatics and RNA blot (Northern) analyses. However, it is not yet known if miRNAs are able to alter gene expression in this class of organisms. We have assessed the possibility that miRNA may mediate circadian regulation of gene expression in the dinoflagellate Lingulodinium polyedrum using the Luciferin Binding Protein (LBP) as a specific example. LBP is a good candidate for regulation by miRNA since mRNA levels are constant over the daily cycle while protein synthesis is restricted by the circadian clock to a period of several hours at the start of the night phase. The transcriptome contains a potential DICER and an ARGONAUTE, suggesting the machinery for generating miRNAs is present. Furthermore, a probe directed against an abundant Symbiodinium miRNA cross reacts on Northern blots. However, L. polyedrum has no small RNAs detectable by ethidium bromide staining, even though higher plant miRNAs run in parallel are readily observed. Illumina sequencing of small RNAs showed that the majority of reads did not have a match in the L. polyedrum transcriptome, and those that did were almost all sense strand mRNA fragments. A direct search for 18–26 nucleotide long RNAs capable of forming duplexes with a 2 base 3’ overhang detected 53 different potential miRNAs, none of which was able to target any of the known circadian regulated genes. Lastly, a microscopy-based test to assess synthesis of the naturally fluorescent LBP in single cells showed that neither double-stranded nor antisense lbp RNA introduced into cells by microparticle bombardment prior to the time of LBP synthesis were able to reduce the amount of LBP produced. Taken together, our results indicate that circadian control of protein synthesis in L. polyedrum is not mediated by miRNAs. PMID:28103286

  8. ZmbZIP91 regulates expression of starch synthesis-related genes by binding to ACTCAT elements in their promoters.

    PubMed

    Chen, Jiang; Yi, Qiang; Cao, Yao; Wei, Bin; Zheng, Lanjie; Xiao, Qianling; Xie, Ying; Gu, Yong; Li, Yangping; Huang, Huanhuan; Wang, Yongbin; Hou, Xianbin; Long, Tiandan; Zhang, Junjie; Liu, Hanmei; Liu, Yinghong; Yu, Guowu; Huang, Yubi

    2016-03-01

    Starch synthesis is a key process that influences crop yield and quality, though little is known about the regulation of this complex metabolic pathway. Here, we present the identification of ZmbZIP91 as a candidate regulator of starch synthesis via co-expression analysis in maize (Zea mays L.). ZmbZIP91 was strongly associated with the expression of starch synthesis genes. Reverse tanscription-PCR (RT-PCR) and RNA in situ hybridization indicated that ZmbZIP91 is highly expressed in maize endosperm, with less expression in leaves. Particle bombardment-mediated transient expression in maize endosperm and leaf protoplasts demonstrated that ZmbZIP91 could positively regulate the expression of starch synthesis genes in both leaves and endosperm. Additionally, the Arabidopsis mutant vip1 carried a mutation in a gene (VIP1) that is homologous to ZmbZIP91, displayed altered growth with less starch in leaves, and ZmbZIP91 was able to complement this phenotype, resulting in normal starch synthesis. A yeast one-hybrid experiment and EMSAs showed that ZmbZIP91 could directly bind to ACTCAT elements in the promoters of starch synthesis genes (pAGPS1, pSSI, pSSIIIa, and pISA1). These results demonstrate that ZmbZIP91 acts as a core regulatory factor in starch synthesis by binding to ACTCAT elements in the promoters of starch synthesis genes.

  9. Akt Phosphorylation and Regulation of Transketolase Is a Nodal Point for Amino Acid Control of Purine Synthesis

    PubMed Central

    Saha, Arindam; Connelly, Stephen; Jiang, Jingjing; Zhuang, Shunhui; Amador, Deron T.; Phan, Tony; Pilz, Renate B.; Boss, Gerry R.

    2014-01-01

    SUMMARY The phosphatidylinositol 3-kinase (PI3K)/Akt pathway integrates environmental clues to regulate cell growth and survival. We showed previously that depriving cells of a single essential amino acid rapidly and reversibly arrests purine synthesis. Here we demonstrate that amino acids via mTORC2 and IκB kinase regulate Akt activity, and Akt association and phosphorylation of transketolase (TKT), a key enzyme of the non-oxidative pentose phosphate pathway (PPP). Akt phosphorylates TKT on Thr382, markedly enhancing enzyme activity and increasing carbon flow through the non-oxidative PPP, thereby increasing purine synthesis. Mice fed a lysine-deficient diet for two days show decreased Akt activity, TKT activity, and purine synthesis in multiple organs. These results provide a new mechanism whereby Akt coordinates amino acid availability with glucose utilization, purine synthesis, and RNA and DNA synthesis. PMID:24981175

  10. Regulation of collagen synthesis in human dermal fibroblasts by ascorbic-induced lipid peroxidation

    SciTech Connect

    Geesin, J.C. Johnson and Johnson Consumer Products, Inc., Skillman, NJ ); Gordon, J.S. ); Gordon, J.S. ); Berg, R.A. )

    1991-03-11

    Ascorbic acid has been shown to stimulate collagen synthesis through the induction of lipid peroxidation which leads to increased transcription of the collagen genes. To test the ability of aldehyde products of lipid peroxidation to mediate this effect, the authors treated cultured fibroblasts with 1-200{mu}M of malondialdehyde, acetaldehyde, glyoxal or hexenal in the presence of lipid peroxidation inducing or noninducing concentrations of ascorbic acid. The treatment process involved either pretreatment of cells for 66hrs with either concentration of ascorbate before a 6hr treatment in the presence of ascorbate and the aldehydes, or 6 or 72hr treatment of the cells in the presence of either concentration of ascorbate plus the aldehydes. No effect of any of these aldehydes was seen on ascorbate-stimulated collagen synthesis. Also, pretreatment of fibroblasts for 24hrs with 100nM phorbol myristate acetate (PMA), which produces down regulation of protein kinase C(PKC), failed to alter the ascorbate-stimulation of collagen synthesis. Additionally, the authors tested the ability of benzamide, a poly ACP ribosylation inhibitor, to inhibit the ascorbate response with no specific effect noted. These results do not support the proposed roles for aldehydes, PKC, or poly ADP ribosylation in the mediation of the lipid peroxidation induced stimulation of collagen synthesis.

  11. Regulation of heat-shock protein synthesis in chicken muscle culture during recovery from heat shock.

    PubMed

    Bag, J

    1983-10-03

    Exposure of chick myotube cultures to a temperature (45 degrees C) higher than their normal growing temperature (37 degrees C) caused extensive synthesis of three major polypeptides of Mr = 25 000, 65 000 and 81 000 referred to as 'heat-shock polypeptides' (hsps). When these cells were allowed to recover from heat-shock treatment at 37 degrees C for 6-8 h, the rate of accumulation of isotope into the 65 000-Mr and 81 000-Mr hsps declined to levels comparable to those in control cultures maintained at 37 degrees C. However, incorporation of isotope in the 25 000-Mr hsp continued at an elevated rate for a longer period than the 65 000-Mr and 81 000-Mr hsps. When heat-shocked cells were allowed to recover at 37 degrees C in the presence of actinomycin D to block new mRNA synthesis, the hsp synthesis as measured by the incorporation of radioactive isotope in these polypeptides continued at levels comparable to those in heat-shocked cells prior to recovery. The block of recovery by actinomycin D was due to the presence of a greater amount of functional hsp mRNAs in the polysomes as compared to untreated controls. The role of competition between the mRNAs for hsps and normal cellular proteins for the translation machinery in regulating protein synthesis during the recovery from heat shock has been discussed.

  12. The Rate-limiting Enzyme in Phosphatidylcholine Synthesis Regulates Proliferation of the Nucleoplasmic ReticulumD⃞

    PubMed Central

    Lagace, Thomas A.; Ridgway, Neale D.

    2005-01-01

    The nucleus contains a network of tubular invaginations of the nuclear envelope (NE), termed the nucleoplasmic reticulum (NR), implicated in transport, gene expression, and calcium homeostasis. Here, we show that proliferation of the NR, measured by the frequency of NE invaginations and tubules, is regulated by CTP:phosphocholine cytidylyltransferase-α (CCTα), the nuclear and rate-limiting enzyme in the CDP–choline pathway for phosphatidylcholine (PtdCho) synthesis. In Chinese hamster ovary (CHO)-K1 cells, fatty acids triggered activation and translocation of CCTα onto intranuclear tubules characteristic of the NR. This was accompanied by a twofold increase in NR tubules quantified by immunostaining for lamin A/C or the NE. CHO MT58 cells expressing a temperature-sensitive CCTα allele displayed reduced PtdCho synthesis and CCTα expression and minimal proliferation of the NR in response to oleate compared with CHO MT58 cells stably expressing CCTα. Expression of CCTα mutants in CHO58 cells revealed that both enzyme activity and membrane binding promoted NR proliferation. In support of a direct role for membrane binding in NR tubule formation, recombinant CCTα caused the deformation of liposomes into tubules in vitro. This demonstrates that a key nuclear enzyme in PtdCho synthesis coordinates lipid synthesis and membrane deformation to promote formation of a dynamic nuclear-cytoplasmic interface. PMID:15635091

  13. Reassessment of the Genetic Regulation of Fatty Acid Synthesis in Escherichia coli: Global Positive Control by the Dual Functional Regulator FadR

    PubMed Central

    My, L.; Ghandour Achkar, N.; Viala, J. P.

    2015-01-01

    ABSTRACT In Escherichia coli, the FadR transcriptional regulator represses the expression of fatty acid degradation (fad) genes. However, FadR is also an activator of the expression of fabA and fabB, two genes involved in unsaturated fatty acid synthesis. Therefore, FadR plays an important role in maintaining the balance between saturated and unsaturated fatty acids in the membrane. We recently showed that FadR also activates the promoter upstream of the fabH gene (L. My, B. Rekoske, J. J. Lemke, J. P. Viala, R. L. Gourse, and E. Bouveret, J Bacteriol 195:3784–3795, 2013, doi:10.1128/JB.00384-13). Furthermore, recent transcriptomic and proteomic data suggested that FadR activates the majority of fatty acid (FA) synthesis genes. In the present study, we tested the role of FadR in the expression of all genes involved in FA synthesis. We found that FadR activates the transcription of all tested FA synthesis genes, and we identified the FadR binding site for each of these genes. This necessitated the reassessment of the transcription start sites for accA and accB genes described previously, and we provide evidence for the presence of multiple promoters driving the expression of these genes. We showed further that regulation by FadR impacts the amount of FA synthesis enzymes in the cell. Our results show that FadR is a global regulator of FA metabolism in E. coli, acting both as a repressor of catabolism and an activator of anabolism, two directly opposing pathways. IMPORTANCE In most bacteria, a transcriptional regulator tunes the level of FA synthesis enzymes. Oddly, such a global regulator still was missing for E. coli, which nonetheless is one of the prominent model bacteria used for engineering biofuel production using the FA synthesis pathway. Our work identifies the FadR functional dual regulator as a global activator of almost all FA synthesis genes in E. coli. Because FadR also is the repressor of FA degradation, FadR acts both as a repressor and an activator

  14. Genome-Wide Association Study Identifies Novel Loci Associated with Circulating Phospho- and Sphingolipid Concentrations

    PubMed Central

    Aulchenko, Yurii S.; Kirichenko, Anatoly V.; Janssens, A. Cecile J. W.; Jansen, Ritsert C.; Gnewuch, Carsten; Domingues, Francisco S.; Pattaro, Cristian; Wild, Sarah H.; Jonasson, Inger; Polasek, Ozren; Zorkoltseva, Irina V.; Hofman, Albert; Karssen, Lennart C.; Struchalin, Maksim; Floyd, James; Igl, Wilmar; Biloglav, Zrinka; Broer, Linda; Pfeufer, Arne; Pichler, Irene; Campbell, Susan; Zaboli, Ghazal; Kolcic, Ivana; Rivadeneira, Fernando; Huffman, Jennifer; Hastie, Nicholas D.; Uitterlinden, Andre; Franke, Lude; Franklin, Christopher S.; Vitart, Veronique; Nelson, Christopher P.; Preuss, Michael; Bis, Joshua C.; O'Donnell, Christopher J.; Franceschini, Nora; Witteman, Jacqueline C. M.; Axenovich, Tatiana; Oostra, Ben A.; Meitinger, Thomas; Hicks, Andrew A.; Hayward, Caroline; Wright, Alan F.; Gyllensten, Ulf; Campbell, Harry; Schmitz, Gerd

    2012-01-01

    Phospho- and sphingolipids are crucial cellular and intracellular compounds. These lipids are required for active transport, a number of enzymatic processes, membrane formation, and cell signalling. Disruption of their metabolism leads to several diseases, with diverse neurological, psychiatric, and metabolic consequences. A large number of phospholipid and sphingolipid species can be detected and measured in human plasma. We conducted a meta-analysis of five European family-based genome-wide association studies (N = 4034) on plasma levels of 24 sphingomyelins (SPM), 9 ceramides (CER), 57 phosphatidylcholines (PC), 20 lysophosphatidylcholines (LPC), 27 phosphatidylethanolamines (PE), and 16 PE-based plasmalogens (PLPE), as well as their proportions in each major class. This effort yielded 25 genome-wide significant loci for phospholipids (smallest P-value = 9.88×10−204) and 10 loci for sphingolipids (smallest P-value = 3.10×10−57). After a correction for multiple comparisons (P-value<2.2×10−9), we observed four novel loci significantly associated with phospholipids (PAQR9, AGPAT1, PKD2L1, PDXDC1) and two with sphingolipids (PLD2 and APOE) explaining up to 3.1% of the variance. Further analysis of the top findings with respect to within class molar proportions uncovered three additional loci for phospholipids (PNLIPRP2, PCDH20, and ABDH3) suggesting their involvement in either fatty acid elongation/saturation processes or fatty acid specific turnover mechanisms. Among those, 14 loci (KCNH7, AGPAT1, PNLIPRP2, SYT9, FADS1-2-3, DLG2, APOA1, ELOVL2, CDK17, LIPC, PDXDC1, PLD2, LASS4, and APOE) mapped into the glycerophospholipid and 12 loci (ILKAP, ITGA9, AGPAT1, FADS1-2-3, APOA1, PCDH20, LIPC, PDXDC1, SGPP1, APOE, LASS4, and PLD2) to the sphingolipid pathways. In large meta-analyses, associations between FADS1-2-3 and carotid intima media thickness, AGPAT1 and type 2 diabetes, and APOA1 and coronary artery disease were observed. In conclusion, our

  15. A Heme-responsive Regulator Controls Synthesis of Staphyloferrin B in Staphylococcus aureus.

    PubMed

    Laakso, Holly A; Marolda, Cristina L; Pinter, Tyler B; Stillman, Martin J; Heinrichs, David E

    2016-01-01

    Staphylococcus aureus possesses a multitude of mechanisms by which it can obtain iron during growth under iron starvation conditions. It expresses an effective heme acquisition system (the iron-regulated surface determinant system), it produces two carboxylate-type siderophores staphyloferrin A and staphyloferrin B (SB), and it expresses transporters for many other siderophores that it does not synthesize. The ferric uptake regulator protein regulates expression of genes encoding all of these systems. Mechanisms of fine-tuning expression of iron-regulated genes, beyond simple iron regulation via ferric uptake regulator, have not been uncovered in this organism. Here, we identify the ninth gene of the sbn operon, sbnI, as encoding a ParB/Spo0J-like protein that is required for expression of genes in the sbn operon from sbnD onward. Expression of sbnD-I is drastically decreased in an sbnI mutant, and the mutant does not synthesize detectable SB during early phases of growth. Thus, SB-mediated iron acquisition is impaired in an sbnI mutant strain. We show that the protein forms dimers and tetramers in solution and binds to DNA within the sbnC coding region. Moreover, we show that SbnI binds heme and that heme-bound SbnI does not bind DNA. Finally, we show that providing exogenous heme to S. aureus growing in an iron-free medium results in delayed synthesis of SB. This is the first study in S. aureus that identifies a DNA-binding regulatory protein that senses heme to control gene expression for siderophore synthesis.

  16. A Heme-responsive Regulator Controls Synthesis of Staphyloferrin B in Staphylococcus aureus*♦

    PubMed Central

    Laakso, Holly A.; Marolda, Cristina L.; Pinter, Tyler B.; Stillman, Martin J.; Heinrichs, David E.

    2016-01-01

    Staphylococcus aureus possesses a multitude of mechanisms by which it can obtain iron during growth under iron starvation conditions. It expresses an effective heme acquisition system (the iron-regulated surface determinant system), it produces two carboxylate-type siderophores staphyloferrin A and staphyloferrin B (SB), and it expresses transporters for many other siderophores that it does not synthesize. The ferric uptake regulator protein regulates expression of genes encoding all of these systems. Mechanisms of fine-tuning expression of iron-regulated genes, beyond simple iron regulation via ferric uptake regulator, have not been uncovered in this organism. Here, we identify the ninth gene of the sbn operon, sbnI, as encoding a ParB/Spo0J-like protein that is required for expression of genes in the sbn operon from sbnD onward. Expression of sbnD–I is drastically decreased in an sbnI mutant, and the mutant does not synthesize detectable SB during early phases of growth. Thus, SB-mediated iron acquisition is impaired in an sbnI mutant strain. We show that the protein forms dimers and tetramers in solution and binds to DNA within the sbnC coding region. Moreover, we show that SbnI binds heme and that heme-bound SbnI does not bind DNA. Finally, we show that providing exogenous heme to S. aureus growing in an iron-free medium results in delayed synthesis of SB. This is the first study in S. aureus that identifies a DNA-binding regulatory protein that senses heme to control gene expression for siderophore synthesis. PMID:26534960

  17. Glycogen synthase kinase-3β positively regulates protein synthesis and cell proliferation through the regulation of translation initiation factor 4E-binding protein 1.

    PubMed

    Shin, S; Wolgamott, L; Tcherkezian, J; Vallabhapurapu, S; Yu, Y; Roux, P P; Yoon, S-O

    2014-03-27

    Protein synthesis has a key role in the control of cell proliferation, and its deregulation is associated with pathological conditions, notably cancer. Rapamycin, an inhibitor of mammalian target of rapamycin complex 1 (mTORC1), was known to inhibit protein synthesis. However, it does not substantially inhibit protein synthesis and cell proliferation in many cancer types. We were interested in finding a novel target in rapamycin-resistant cancer. The rate-limiting factor for translation is eukaryotic translation initiation factor 4E (eIF4E), which is negatively regulated by eIF4E-binding protein 1 (4E-BP1). Here, we provide evidence that glycogen synthase kinase (GSK)-3β promotes cell proliferation through positive regulation of protein synthesis. We found that GSK-3β phosphorylates and inactivates 4E-BP1, thereby increasing eIF4E-dependent protein synthesis. Considering the clinical relevance of pathways regulating protein synthesis, our study provides a promising new strategy and target for cancer therapy.

  18. DNA polymerase-α regulates type I interferon activation through cytosolic RNA:DNA synthesis

    PubMed Central

    Starokadomskyy, Petro; Gemelli, Terry; Rios, Jonathan J.; Xing, Chao; Wang, Richard C.; Li, Haiying; Pokatayev, Vladislav; Dozmorov, Igor; Khan, Shaheen; Miyata, Naoteru; Fraile, Guadalupe; Raj, Prithvi; Xu, Zhe; Xu, Zigang; Ma, Lin; Lin, Zhimiao; Wang, Huijun; Yang, Yong; Ben-Amitai, Dan; Orenstein, Naama; Mussaffi, Huda; Baselga, Eulalia; Tadini, Gianluca; Grunebaum, Eyal; Sarajlija, Adrijan; Krzewski, Konrad; Wakeland, Edward K.; Yan, Nan; de la Morena, Maria Teresa; Zinn, Andrew R.; Burstein, Ezra

    2016-01-01

    Aberrant nucleic acids generated during viral replication are the main trigger for antiviral immunity, and mutations disrupting nucleic acid metabolism can lead to autoinflammatory disorders. Here we investigated the etiology of X-linked reticulate pigmentary disorder (XLPDR), a primary immunodeficiency with autoinflammatory features. We discovered that XLPDR is caused by an intronic mutation that disrupts expression of POLA1, the gene encoding the catalytic subunit of DNA polymerase-α. Unexpectedly, POLA1 deficiency results in increased type I interferon production. This enzyme is necessary for RNA:DNA primer synthesis during DNA replication and strikingly, POLA1 is also required for the synthesis of cytosolic RNA:DNA, which directly modulates interferon activation. Altogether, this work identified POLA1 as a critical regulator of the type I interferon response. PMID:27019227

  19. Down regulation of gene related sex hormone synthesis pathway in mouse testes by miroestrol and deoxymiroestrol.

    PubMed

    Udomsuk, Latiporn; Juengwatanatrakul, Thaweesak; Putalun, Waraporn; Jarukamjorn, Kanokwan

    2011-12-01

    Miroestrol and deoxymiroestrol are phytoestrogens isolated from tuberous root of Pueraria candollei var. mirifica. Modulatory effects of miroestrol and deoxymiroestrol on enzymes involved in sex-hormone synthesis pathway in male C57BL/6 mice were investigated using semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). Miroestrol and deoxymiroestrol suppressed the expressions of 3β-HSD, 17β-HSD1, and CYP17 while CYP19 mRNA expression was slightly decreased. In addition, the expression of 17β-HSD2 was induced in correlation with those did by estradiol. These observations supported that miroestrol and deoxymiroestrol could exhibit the same effect as estradiol regarding regulation of testicular gene related sex hormone synthesis pathway.

  20. Three-dimensional imaging of cholesterol and sphingolipids within a Madin-Darby canine kidney cell

    SciTech Connect

    Yeager, Ashley N.; Weber, Peter K.; Kraft, Mary L.

    2016-01-08

    Metabolic stable isotope incorporation and secondary ion mass spectrometry(SIMS) depth profiling performed on a Cameca NanoSIMS 50 were used to image the 18O-cholesterol and 15N-sphingolipid distributions within a portion of a Madin-Darby canine kidney (MDCK) cell. Three-dimensional representations of the component-specific isotope distributions show clearly defined regions of 18O-cholesterol and 15N-sphingolipid enrichment that seem to be separate subcellular compartments. Furthermore, the low levels of nitrogen-containing secondary ions detected at the 18O-enriched regions suggest that these 18O-cholesterol-rich structures may be lipiddroplets, which have a core consisting of cholesterol esters and triacylglycerides.

  1. Three-dimensional imaging of cholesterol and sphingolipids within a Madin-Darby canine kidney cell

    DOE PAGES

    Yeager, Ashley N.; Weber, Peter K.; Kraft, Mary L.

    2016-01-08

    Metabolic stable isotope incorporation and secondary ion mass spectrometry(SIMS) depth profiling performed on a Cameca NanoSIMS 50 were used to image the 18O-cholesterol and 15N-sphingolipid distributions within a portion of a Madin-Darby canine kidney (MDCK) cell. Three-dimensional representations of the component-specific isotope distributions show clearly defined regions of 18O-cholesterol and 15N-sphingolipid enrichment that seem to be separate subcellular compartments. Furthermore, the low levels of nitrogen-containing secondary ions detected at the 18O-enriched regions suggest that these 18O-cholesterol-rich structures may be lipiddroplets, which have a core consisting of cholesterol esters and triacylglycerides.

  2. Free-radical Destruction of Sphingolipids Resulting in 2-hexadecenal Formation

    PubMed Central

    Shadyro, Oleg; Lisovskaya, Alexandra; Semenkova, Galina; Edimecheva, Irina; Amaegberi, Nadezda

    2015-01-01

    The action of hypochlorous acid (HOCl) and γ-radiation on aqueous lysosphingolipid dispersions was found to produce 2-hexadecenal (Hex). This process includes the stages of formation of nitrogen-centered radicals from the starting molecules and the subsequent fragmentation of these radicals via the rupture of C–C and O–H bonds. These findings prove the existence of a nonenzymatic pathway of sphingolipid destruction leading to the formation of Hex, which possesses a wide spectrum of biological activity. Analysis of the effect of HOCl on transplantable rat glioma C6 cells and human embryonic kidney 293 cells points to the formation of Hex. This suggests that the described mechanism of free-radical destruction of sphingolipids may be replicated on cell culture under the stress of active chlorine forms. PMID:25861222

  3. Segregation of sphingolipids and sterols during formation of secretory vesicles at the trans-Golgi network

    PubMed Central

    Klemm, Robin W.; Ejsing, Christer S.; Surma, Michal A.; Kaiser, Hermann-Josef; Gerl, Mathias J.; Sampaio, Julio L.; de Robillard, Quentin; Ferguson, Charles; Proszynski, Tomasz J.; Shevchenko, Andrej

    2009-01-01

    The trans-Golgi network (TGN) is the major sorting station in the secretory pathway of all eukaryotic cells. How the TGN sorts proteins and lipids to generate the enrichment of sphingolipids and sterols at the plasma membrane is poorly understood. To address this fundamental question in membrane trafficking, we devised an immunoisolation procedure for specific recovery of post-Golgi secretory vesicles transporting a transmembrane raft protein from the TGN to the cell surface in the yeast Saccharomyces cerevisiae. Using a novel quantitative shotgun lipidomics approach, we could demonstrate that TGN sorting selectively enriched ergosterol and sphingolipid species in the immunoisolated secretory vesicles. This finding, for the first time, indicates that the TGN exhibits the capacity to sort membrane lipids. Furthermore, the observation that the immunoisolated vesicles exhibited a higher membrane order than the late Golgi membrane, as measured by C-Laurdan spectrophotometry, strongly suggests that lipid rafts play a role in the TGN-sorting machinery. PMID:19433450

  4. Neuronal GPCR OCTR-1 regulates innate immunity by controlling protein synthesis in Caenorhabditis elegans

    PubMed Central

    Liu, Yiyong; Sellegounder, Durai; Sun, Jingru

    2016-01-01

    Upon pathogen infection, microbial killing pathways and cellular stress pathways are rapidly activated by the host innate immune system. These pathways must be tightly regulated because insufficient or excessive immune responses have deleterious consequences. Increasing evidence indicates that the nervous system regulates the immune system to confer coordinated protection to the host. However, the precise mechanisms of neural-immune communication remain unclear. Previously we have demonstrated that OCTR-1, a neuronal G protein-coupled receptor, functions in the sensory neurons ASH and ASI to suppress innate immune responses in non-neural tissues against Pseudomonas aeruginosa in Caenorhabditis elegans. In the current study, by using a mass spectrometry-based quantitative proteomics approach, we discovered that OCTR-1 regulates innate immunity by suppressing translation and the unfolded protein response (UPR) pathways at the protein level. Functional assays revealed that OCTR-1 inhibits specific protein synthesis factors such as ribosomal protein RPS-1 and translation initiation factor EIF-3.J to reduce infection-triggered protein synthesis and UPR. Translational inhibition by chemicals abolishes the OCTR-1-controlled innate immune responses, indicating that activation of the OCTR-1 pathway is dependent on translation upregulation such as that induced by pathogen infection. Because OCTR-1 downregulates protein translation activities, the OCTR-1 pathway could function to suppress excessive responses to infection or to restore protein homeostasis after infection. PMID:27833098

  5. Sphingolipid Metabolism Correlates with Cerebrospinal Fluid Beta Amyloid Levels in Alzheimer’s Disease

    PubMed Central

    Fonteh, Alfred N.; Ormseth, Cora; Chiang, Jiarong; Cipolla, Matthew; Arakaki, Xianghong; Harrington, Michael G.

    2015-01-01

    Sphingolipids are important in many brain functions but their role in Alzheimer’s disease (AD) is not completely defined. A major limit is availability of fresh brain tissue with defined AD pathology. The discovery that cerebrospinal fluid (CSF) contains abundant nanoparticles that include synaptic vesicles and large dense core vesicles offer an accessible sample to study these organelles, while the supernatant fluid allows study of brain interstitial metabolism. Our objective was to characterize sphingolipids in nanoparticles representative of membrane vesicle metabolism, and in supernatant fluid representative of interstitial metabolism from study participants with varying levels of cognitive dysfunction. We recently described the recruitment, diagnosis, and CSF collection from cognitively normal or impaired study participants. Using liquid chromatography tandem mass spectrometry, we report that cognitively normal participants had measureable levels of sphingomyelin, ceramide, and dihydroceramide species, but that their distribution differed between nanoparticles and supernatant fluid, and further differed in those with cognitive impairment. In CSF from AD compared with cognitively normal participants: a) total sphingomyelin levels were lower in nanoparticles and supernatant fluid; b) levels of ceramide species were lower in nanoparticles and higher in supernatant fluid; c) three sphingomyelin species were reduced in the nanoparticle fraction. Moreover, three sphingomyelin species in the nanoparticle fraction were lower in mild cognitive impairment compared with cognitively normal participants. The activity of acid, but not neutral sphingomyelinase was significantly reduced in the CSF from AD participants. The reduction in acid sphingomylinase in CSF from AD participants was independent of depression and psychotropic medications. Acid sphingomyelinase activity positively correlated with amyloid β42 concentration in CSF from cognitively normal but not impaired

  6. Modifications to the translational apparatus which affect the regulation of protein synthesis in sea urchin embryos

    SciTech Connect

    Scalise, F.W.

    1988-01-01

    Protein synthesis can be regulated at a number of cellular levels. I have examined how modifications to specific components of the protein synthetic machinery are involved in regulating the efficiency of initiation of translation during early sea urchin embryogenesis. It is demonstrated that Ca{sup 2+} concentrations exceeding 500 uM cause the inhibition of protein synthesis in cell-free translation lysates prepared from sea urchin embryos. Specific changes in the state of phosphorylation of at least 8 proteins occur during this Ca{sup 2+}-mediated repression of translation. Analysis of these proteins has indicated that, unlike mammalian systems, there is no detectable level of Ca{sup 2+}-dependent phosphorylation of the {alpha}subunit eIF-2. Two of the proteins which do become phosphorylated in response to Ca{sup 2+} are calmodulin and an isoelectric form of sea urchin eIF-4D. In addition, 2 proteins which share similarities with kinases involved in the regulation of protein synthesis in mammalian cells, also become phosphorylated. I have investigated the consequences of changes in eIF-4D during sea urchin embryogenesis because it has been proposed that a polyamine-mediated conversion of lysine to hypusine in this factor may enhance translational activity. It is demonstrated that ({sup 3}H) spermidine-derived radioactivity is incorporated into a number of proteins when sea urchin embryos are labeled in vivo, and that the pattern of individual proteins that become labeled changes over the course of the first 30 hr of development.

  7. Ankyrin repeat and SOCS box protein 15 regulates protein synthesis in skeletal muscle.

    PubMed

    McDaneld, T G; Hannon, K; Moody, D E

    2006-06-01

    Ankyrin repeat and SOCS box protein 15 (ASB15) is an Asb family member expressed predominantly in skeletal muscle. We have previously reported that ASB15 mRNA abundance decreases after administration of beta-adrenergic receptor agonists. Because beta-adrenergic receptor agonists are known to stimulate muscle hypertrophy, the objective of this study was to determine whether ASB15 regulates cellular processes that contribute to muscle growth. Stable myoblast C2C12 cells expressing full-length ASB15 (ASB15-FL) and ASB15 lacking the ankyrin repeat (ASB15-Ank) or SOCS box (ASB15-SOCS) motifs were evaluated for changes in proliferation, differentiation, protein synthesis, and protein degradation. Expression of ASB15-FL caused a delay in differentiation, followed by an increase in protein synthesis of approximately 34% (P<0.05). A consistent effect of ASB15 overexpression was observed in vivo, where ectopic expression of ASB15 increased skeletal muscle fiber area (P<0.0001) after 9 days. Expression of ASB15-SOCS altered differentiation of myoblasts, resulting in detachment of cells from culture plates. Expression of ASB15-Ank increased protein degradation by 84 h of differentiation (P<0.05), and in vivo ectopic expression of an ASB15 construct lacking both the ankyrin repeat and SOCS box motifs decreased skeletal muscle fiber area (P<0.0001). Together, these results suggest ASB15 participates in the regulation of protein turnover and muscle cell development by stimulating protein synthesis and regulating differentiation of muscle cells. This is the first study to demonstrate a role for an Asb family member in skeletal muscle growth.

  8. Eukaryotic elongation factor 2 kinase regulates the synthesis of microtubule-related proteins in neurons.

    PubMed

    Kenney, Justin W; Genheden, Maja; Moon, Kyung-Mee; Wang, Xuemin; Foster, Leonard J; Proud, Christopher G

    2016-01-01

    Modulation of the elongation phase of protein synthesis is important for numerous physiological processes in both neurons and other cell types. Elongation is primarily regulated via eukaryotic elongation factor 2 kinase (eEF2K). However, the consequence of altering eEF2K activity on the synthesis of specific proteins is largely unknown. Using both pharmacological and genetic manipulations of eEF2K combined with two protein-labeling techniques, stable isotope labeling of amino acids in cell culture and bio-orthogonal non-canonical amino acid tagging, we identified a subset of proteins whose synthesis is sensitive to inhibition of eEF2K in murine primary cortical neurons. Gene ontology (GO) analyses indicated that processes related to microtubules are particularly sensitive to eEF2K inhibition. Our findings suggest that eEF2K likely contributes to neuronal function by regulating the synthesis of microtubule-related proteins. Modulation of the elongation phase of protein synthesis is important for numerous physiological processes in neurons. Here, using labeling of new proteins coupled with proteomic techniques in primary cortical neurons, we find that the synthesis of microtubule-related proteins is up-regulated by inhibition of elongation. This suggests that translation elongation is a key regulator of cytoskeletal dynamics in neurons.

  9. Additional nitrogen fertilization at heading time of rice down-regulates cellulose synthesis in seed endosperm.

    PubMed

    Midorikawa, Keiko; Kuroda, Masaharu; Terauchi, Kaede; Hoshi, Masako; Ikenaga, Sachiko; Ishimaru, Yoshiro; Abe, Keiko; Asakura, Tomiko

    2014-01-01

    The balance between carbon and nitrogen is a key determinant of seed storage components, and thus, is of great importance to rice and other seed-based food crops. To clarify the influence of the rhizosphere carbon/nitrogen balance during the maturation stage of several seed components, transcriptome analysis was performed on the seeds from rice plants that were provided additional nitrogen fertilization at heading time. As a result, it was assessed that genes associated with molecular processes such as photosynthesis, trehalose metabolism, carbon fixation, amino acid metabolism, and cell wall metabolism were differentially expressed. Moreover, cellulose and sucrose synthases, which are involved in cellulose synthesis, were down-regulated. Therefore, we compared cellulose content of mature seeds that were treated with additional nitrogen fertilization with those from control plants using calcofluor staining. In these experiments, cellulose content in endosperm from plants receiving additional nitrogen fertilization was less than that in control endosperm. Other starch synthesis-related genes such as starch synthase 1, starch phosphorylase 2, and branching enzyme 3 were also down-regulated, whereas some α-amylase and β-amylase genes were up-regulated. On the other hand, mRNA expression of amino acid biosynthesis-related molecules was up-regulated. Moreover, additional nitrogen fertilization caused accumulation of storage proteins and up-regulated Cys-poor prolamin mRNA expression. These data suggest that additional nitrogen fertilization at heading time changes the expression of some storage substance-related genes and reduces cellulose levels in endosperm.

  10. Rapid nanoscale quantitative analysis of plant sphingolipid long-chain bases by GC-MS.

    PubMed

    Cacas, Jean-Luc; Melser, Su; Domergue, Frédéric; Joubès, Jérôme; Bourdenx, Brice; Schmitter, Jean-Marie; Mongrand, Sébastien

    2012-07-01

    In eukaryotic organisms, sphingolipids are major structural lipids of biological membranes and perform additional essential functions as signalling molecules. While long-chain bases (LCB), the common precursor to all sphingolipid classes, is represented by only one major molecular species in animals and fungi, up to nine LCB have been found in plants. In the absence of genuine plant sphingolipid references required for proper quantification, we have reinvestigated and optimized a protocol destined to the quantification of total plant LCB that relies on the use of gas chromatography-mass spectrometry (GC-MS). This rapid three-step protocol sequentially involves (1) the release of LCB from biological samples using barium hydroxide solution, (2) their oxidation into aldehydes by metaperiodate, and (3) the subsequent identification/quantification of these aldehydes by GC-MS. It is simple and reliable and enables separation of aldehydes upon their stero-specificity. It further enables the quantification of total LCB from a wide variety of samples including yeast and animal cell cultures.

  11. PLP-dependent enzymes as entry and exit gates of sphingolipid metabolism

    PubMed Central

    Bourquin, Florence; Capitani, Guido; Grütter, Markus Gerhard

    2011-01-01

    Sphingolipids are membrane constituents as well as signaling molecules involved in many essential cellular processes. Serine palmitoyltransferase (SPT) and sphingosine-1-phosphate lyase (SPL), both PLP (pyridoxal 5′-phosphate)-dependent enzymes, function as entry and exit gates of the sphingolipid metabolism. SPT catalyzes the condensation of serine and a fatty acid into 3-keto-dihydrosphingosine, whereas SPL degrades sphingosine-1-phosphate (S1P) into phosphoethanolamine and a long-chain aldehyde. The recently solved X-ray structures of prokaryotic homologs of SPT and SPL combined with functional studies provide insight into the structure–function relationship of the two enzymes. Despite carrying out different reactions, the two enzymes reveal striking similarities in the overall fold, topology, and residues crucial for activity. Unlike their eukaryotic counterparts, bacterial SPT and SPL lack a transmembrane helix, making them targets of choice for biochemical characterization because the use of detergents can be avoided. Both human enzymes are linked to severe diseases or disorders and might therefore serve as targets for the development of therapeutics aiming at the modulation of their activity. This review gives an overview of the sphingolipid metabolism and of the available biochemical studies of prokaryotic SPT and SPL, and discusses the major similarities and differences to the corresponding eukaryotic enzymes. PMID:21710479

  12. GC/MS-based metabolomic studies reveal key roles of glycine in regulating silk synthesis in silkworm, Bombyx mori.

    PubMed

    Chen, Quanmei; Liu, Xinyu; Zhao, Ping; Sun, Yanhui; Zhao, Xinjie; Xiong, Ying; Xu, Guowang; Xia, Qingyou

    2015-02-01

    Metabolic profiling of silkworm, especially the factors that affect silk synthesis at the metabolic level, is little known. Herein, metabolomic method based on gas chromatography-mass spectrometry was applied to identify key metabolic changes in silk synthesis deficient silkworms. Forty-six differential metabolites were identified in Nd group with the defect of silk synthesis. Significant changes in the levels of glycine and uric acid (up-regulation), carbohydrates and free fatty acids (down-regulation) were observed. The further metabolomics of silk synthesis deficient silkworms by decreasing silk proteins synthesis using knocking out fibroin heavy chain gene or extirpating silk glands operation showed that the changes of the metabolites were almost consistent with those of the Nd group. Furthermore, the increased silk yields by supplying more glycine or its related metabolite confirmed that glycine is a key metabolite to regulate silk synthesis. These findings provide important insights into the regulation between metabolic profiling and silk synthesis.

  13. Leucine is a major regulator of muscle protein synthesis in neonates

    PubMed Central

    Columbus, Daniel A; Fiorotto, Marta L; Davis, Teresa A

    2014-01-01

    Approximately 10% of infants born in the United States are of low birth weight. Growth failure during the neonatal period is a common occurrence in low birth weight infants due to their inability to tolerate full feeds, concerns about advancing amino acid supply, and high nutrient requirements for growth. An improved understanding of the nutritional regulation of growth during this critical period of development is vital for the development of strategies to improve lean growth. Past studies with animal models have demonstrated that muscle protein synthesis is increased substantially following a meal and that this increase is due to the postprandial rise in amino acids as well as insulin, which independently stimulate protein synthesis in a mammalian target of rapamycin (mTOR)-dependent manner. Further studies have elucidated that leucine, in particular, as well as its metabolites, α-ketoisocaproic acid and β-hydroxy-β-methylbutyrate, have unique anabolic properties. Supplementation with leucine, provided either parenterally or enterally, has been shown to enhance muscle protein synthesis in neonatal pigs, making it an ideal candidate for stimulating growth of low birth weight infants. PMID:25408462

  14. Regulation of yeast DNA polymerase δ-mediated strand displacement synthesis by 5'-flaps.

    PubMed

    Koc, Katrina N; Stodola, Joseph L; Burgers, Peter M; Galletto, Roberto

    2015-04-30

    The strand displacement activity of DNA polymerase δ is strongly stimulated by its interaction with proliferating cell nuclear antigen (PCNA). However, inactivation of the 3'-5' exonuclease activity is sufficient to allow the polymerase to carry out strand displacement even in the absence of PCNA. We have examined in vitro the basic biochemical properties that allow Pol δ-exo(-) to carry out strand displacement synthesis and discovered that it is regulated by the 5'-flaps in the DNA strand to be displaced. Under conditions where Pol δ carries out strand displacement synthesis, the presence of long 5'-flaps or addition in trans of ssDNA suppress this activity. This suggests the presence of a secondary DNA binding site on the enzyme that is responsible for modulation of strand displacement activity. The inhibitory effect of a long 5'-flap can be suppressed by its interaction with single-stranded DNA binding proteins. However, this relief of flap-inhibition does not simply originate from binding of Replication Protein A to the flap and sequestering it. Interaction of Pol δ with PCNA eliminates flap-mediated inhibition of strand displacement synthesis by masking the secondary DNA site on the polymerase. These data suggest that in addition to enhancing the processivity of the polymerase PCNA is an allosteric modulator of other Pol δ activities.

  15. Synthesis, Delivery and Regulation of Eukaryotic Heme and Fe-S Cluster Cofactors

    PubMed Central

    Barupala, Dulmini P.; Dzul, Stephen P.; Riggs-Gelasco, Pamela Jo; Stemmler, Timothy L.

    2016-01-01

    In humans, the bulk of iron in the body (over 75%) is directed towards heme- or Fe-S cluster cofactor synthesis, and the complex, highly regulated pathways in place to accomplish biosynthesis have evolved to safely assemble and load these cofactors into apoprotein partners. In eukaryotes, heme biosynthesis is both initiated and finalized within the mitochondria, while cellular Fe-S cluster assembly is controlled by correlated pathways both within the mitochondria and within the cytosol. Iron plays a vital role in a wide array of metabolic processes and defects in iron cofactor assembly leads to human diseases. This review describes progress towards our molecular-level understanding of cellular heme and Fe-S cluster biosynthesis, focusing on the regulation and mechanistic details that are essential for understanding human disorders related to the breakdown in these essential pathways. PMID:26785297

  16. GAD67-mediated GABA synthesis and signaling regulate inhibitory synaptic innervation in the visual cortex.

    PubMed

    Chattopadhyaya, Bidisha; Di Cristo, Graziella; Wu, Cai Zhi; Knott, Graham; Kuhlman, Sandra; Fu, Yu; Palmiter, Richard D; Huang, Z Josh

    2007-06-21

    The development of GABAergic inhibitory circuits is shaped by neural activity, but the underlying mechanisms are unclear. Here, we demonstrate a novel function of GABA in regulating GABAergic innervation in the adolescent brain, when GABA is mainly known as an inhibitory transmitter. Conditional knockdown of the rate-limiting synthetic enzyme GAD67 in basket interneurons in adolescent visual cortex resulted in cell autonomous deficits in axon branching, perisomatic synapse formation around pyramidal neurons, and complexity of the innervation fields; the same manipulation had little influence on the subsequent maintenance of perisomatic synapses. These effects of GABA deficiency were rescued by suppressing GABA reuptake and by GABA receptor agonists. Germline knockdown of GAD67 but not GAD65 showed similar deficits, suggesting a specific role of GAD67 in the maturation of perisomatic innervation. Since intracellular GABA levels are modulated by neuronal activity, our results implicate GAD67-mediated GABA synthesis in activity-dependent regulation of inhibitory innervation patterns.

  17. Dipeptides Inhibit Melanin Synthesis in Mel-Ab Cells through Down-Regulation of Tyrosinase

    PubMed Central

    Lee, Hyun-e; Kim, Eun-Hyun; Choi, Hye-Ryung; Sohn, Uy Dong; Yun, Hye-Young; Baek, Kwang Jin; Kwon, Nyoun Soo; Park, Kyoung-Chan

    2012-01-01

    This study investigated the effects of proline-serine (PS) and valine-serine (VS) dipeptides on melanogenesis in Mel-Ab cells. Proline-serine and VS significantly inhibited melanin synthesis in a concentration-dependent manner, though neither dipeptide directly inhibited tyrosinase activity in a cell-free system. Both PS and VS down-regulated the expression of microphthalmia-associated transcription factor (MITF) and tyrosinase. In a follow-up study also described here, the effects of these dipeptides on melanogenesis-related signal transduction were quantified. Specifically, PS and VS induced ERK phosphorylation, though they had no effect on phosphorylation of the cAMP response element binding protein (CREB). These data suggest that PS and VS inhibit melanogenesis through ERK phosphorylation and subsequent down-regulation of MITF and tyrosinase. Properties of these dipeptides are compatible with application as skin-whitening agents. PMID:22915995

  18. [The first steps of chlorophyll synthesis: RNA involvement and regulation]. Progress report, January 1990--June 1992

    SciTech Connect

    Soell, D.

    1992-12-31

    Glu-tRNA{sup Glu} is synthesized from glutamate and tRNA{sup Glu} by glutamyl-tRNA synthetase (GluRS). Recent work has demonstrated that Glu-tRNA{sup Glu} has dual functions and is a precursor for protein and 5-aminolevulinate (ALA) synthesis. Current data does not provide compelling evidence for the notion that GluRS is regulated by chlorophyll precursors or in concert with the other enzymes of ALA synthesis. We have redefined the C5-pathway as a two-step route to ALA starting with Glu-tRNA{sup Glu}. Only two enzymes, Glu-tRNA reductase (GluTR) and GSA-2,1-amino-mutase (GSA-AM), are specifically involved in ALA synthesis. We have purified these enzymatic activities from Chlamydomonas and demonstrated that the two purified proteins in the presence of their cofactors NADPH and pyridoxal phosphate are sufficient for the in vitro Glu-tRNA {yields} ALA conversion. We have cloned the genes encoding GluTR. The sequences of the GluTR proteins deduced from these genes share highly conserved regions with those of bacterial origin. We havealso cloned and analyzed the gene encoding GSA-AM from Arabidopsis. As in Salmonella typhimurium, there are indications of the existence of an additional pathway for ALA formation in E. coli. To shed light on the recognition of the single tRNA{sup Glu} by the chloroplast enzymes GluTR, GluRS we characterized a chlorophyll-deficient mutant of Euglena having tRNA{sup Glu} with a point mutation in the T{Psi}C-loop. The altered tRNA supports protein but not ALA synthesis.

  19. Regulation of chitin synthesis in the larval midgut of Manduca sexta.

    PubMed

    Zimoch, L; Hogenkamp, D G; Kramer, K J; Muthukrishnan, S; Merzendorfer, H

    2005-06-01

    In insects, chitin is not only synthesized by ectodermal cells that form chitinous cuticles, but also by endodermal cells of the midgut that secrete a chitinous peritrophic matrix. Using anti-chitin synthase (CHS) antibodies, we previously demonstrated that in the midgut of Manduca sexta, CHS is expressed by two cell types, tracheal cells forming a basal tracheal network and columnar cells forming the apical brush border [Zimoch and Merzendorfer, 2002, Cell Tissue Res. 308, 287-297]. Now, we show that two different genes, MsCHS1 and MsCHS2, encode CHSs of midgut tracheae and columnar cells, respectively. To investigate MsCHS2 expression and activity in the course of the larval development, we monitored chitin synthesis, enzyme levels as well as mRNA amounts. All of the tested parameters were significantly reduced during molting and in the wandering stage when compared to the values obtained from intermolt feeding larvae. By contrast, MsCHS1 appeared to be inversely regulated because its mRNA was detectable only during the molt at the time when tracheal growth occurs at the basal site of the midgut. To further examine midgut chitin synthesis, we measured enzyme activity in crude midgut extracts and different membrane fractions. When we analysed trypsin-mediated proteolytic activation, a phenomenon previously reported for insect and fungal systems, we recognized that midgut chitin synthesis was only activated in crude extracts, but not in the 12,000 g membrane fraction. However, proteolytic activation by trypsin in the 12,000 g membrane fraction could be reconstituted by re-adding a soluble fraction, indicating that limited proteolysis affects an unknown soluble factor, a process that in turn activates chitin synthesis.

  20. Regulation of thiamine synthesis in Saccharomyces cerevisiae for improved pyruvate production.

    PubMed

    Xu, Guoqiang; Hua, Qiang; Duan, Ningjun; Liu, Liming; Chen, Jian

    2012-06-01

    Metabolic engineering of Saccharomyces cerevisiae for high-yield production of carboxylic acid requires a cytosolic pyruvate pool as precursor. In this study, a novel strategy to improve pyruvate production and reduce metabolic by-products via regulating thiamine synthesis was explored. Two of the thiamine biosynthesis regulatory genes, THI2 and THI3, were disrupted in the S. cerevisiae parent strain FMME-002. The mutants FMME-002ΔTHI2 and FMME-002ΔTHI3 both exhibited an enhanced pyruvate yield. Moreover, FMME-002ΔTHI2 achieved a relatively higher pyruvate production, and the highest concentration of pyruvate was achieved when 0.04 µ m thiamine was added. Enzyme assays and fermentation profiles of the THI2-complemented strain indicated that the observed metabolic changes represented intrinsic effects of THI2 deletion on the physiology of S. cerevisiae. Under optimal C:N ratio conditions, FMME-002ΔTHI2 produced pyruvate up to 8.21 ± 0.30 g/l, whereas the ethanol titre decreased to 2.21 ± 0.24 g/l after 96 h of cultivation. These results demonstrate the possibility of improving pyruvate production by regulating thiamine synthesis in S. cerevisiae.

  1. Modulation of xylosyltransferase I expression provides a mechanism regulating glycosaminoglycan chain synthesis during cartilage destruction and repair.

    PubMed

    Venkatesan, Narayanan; Barré, Lydia; Magdalou, Jacques; Mainard, Didier; Netter, Patrick; Fournel-Gigleux, Sylvie; Ouzzine, Mohamed

    2009-03-01

    Osteoarthritis and rheumatoid arthritis are characterized by loss of proteoglycans (PGs) and their glycosaminoglycan (GAG) chains that are essential for cartilage function. Here, we investigated the role of glycosyltransferases (GTs) responsible for PG-GAG chain assembly during joint cartilage destruction and repair processes. At various times after antigen-induced arthritis (AIA) and papain-induced cartilage repair in rats, PG synthesis and deposition, expression of GTs, and GAG chain composition were analyzed. Our data showed that expression of the GT xylosyltransferase I (XT-I) gene initiating PG-GAG chain synthesis was significantly reduced in AIA rat cartilage and was associated with a decrease in PG synthesis. Interestingly, interleukin-1beta, the main proinflammatory cytokine incriminated in joint diseases, down-regulated the XT-I gene expression with a concomitant decrease in PG synthesis in rat cartilage explants ex vivo. However, cartilage from papain-injected rat knees showed up-regulation of XT-I gene expression and increased PG synthesis at early stages of cartilage repair, a process associated with up-regulation of TGF-beta1 gene expression and mediated by p38 mitogen-activated protein kinase activation. Consistently, silencing of XT-I expression by intraarticular injection of XT-I shRNA in rat knees prevented cartilage repair by decreasing PG synthesis and content. These findings show that GTs play a key role in the loss of PG-GAGs in joint diseases and identify novel targets for stimulating cartilage repair.

  2. Pseudomonas syringae infection triggers de novo synthesis of phytosphingosine from sphinganine in Arabidopsis thaliana.

    PubMed

    Peer, Markus; Stegmann, Martin; Mueller, Martin J; Waller, Frank

    2010-09-24

    Sphingolipids are important membrane components and also regulate cell proliferation and apoptosis. We detected a fast increase of the free sphingobase t18:0 (phytosphinganine) in Arabidopsis leaves after inoculation with an avirulent strain of the bacterial pathogen Pseudomonas syringae pathovar tomato, characterized by host cell death reactions. The induction of phytosphinganine was more transient in virulent interactions lacking cell death reactions, suggesting a positive role of t18:0 in the plants' response to pathogens, e.g. the hypersensitive response. In the mutant sphingobase hydroxylase 1 (sbh1-1), Pseudomonas induced elevated free d18:0 levels. As total t18:0 contents (after hydrolysis of ceramides) were not reduced in sbh1-1, the pathogen-triggered t18:0 increase most likely results from de novo synthesis from d18:0 which would require SBH1.

  3. Melatonin Regulates Root Meristem by Repressing Auxin Synthesis and Polar Auxin Transport in Arabidopsis

    PubMed Central

    Wang, Qiannan; An, Bang; Wei, Yunxie; Reiter, Russel J.; Shi, Haitao; Luo, Hongli; He, Chaozu

    2016-01-01

    Melatonin (N-acetyl-5-methoxytryptamine) plays important roles in regulating both biotic and abiotic stress tolerance, biological rhythms, plant growth and development. Sharing the same substrate (tryptophan) for the biosynthesis, melatonin and auxin also have similar effects in plant development. However, the specific function of melatonin in modulating plant root growth and the relationship between melatonin and auxin as well as underlying mechanisms are still unclear. In this study, we found high concentration of melatonin remarkably inhibited root growth in Arabidopsis by reducing root meristem size. Further studies showed that melatonin negatively regulated auxin biosynthesis, the expression of PINFORMED (PIN) proteins as well as auxin response in Arabidopsis. Moreover, the root growth of the triple mutant pin1pin3pin7 was more tolerant than that of wild-type in response to melatonin treatment, suggesting the essential role of PIN1/3/7 in melatonin-mediated root growth. Combination treatment of melatonin and 5-Triiodobenzoic acid (TIBA) did not enhance melatonin-mediated reduction of root meristem size, indicating that polar auxin transport (PAT) may be necessary for the regulation of root meristem size by melatonin treatment. Taken together, this study indicates that melatonin regulates root growth in Arabidopsis, through auxin synthesis and polar auxin transport, at least partially. PMID:28018411

  4. Phosphatase control of 4E-BP1 phosphorylation state is central for glycolytic regulation of retinal protein synthesis.

    PubMed

    Gardner, Thomas W; Abcouwer, Steven F; Losiewicz, Mandy K; Fort, Patrice E

    2015-09-15

    Control of protein synthesis in insulin-responsive tissues has been well characterized, but relatively little is known about how this process is regulated in nervous tissues. The retina exhibits a relatively high protein synthesis rate, coinciding with high basal Akt and metabolic activities, with the majority of retinal ATP being derived from aerobic glycolysis. We examined the dependency of retinal protein synthesis on the Akt-mTOR signaling and glycolysis using ex vivo rat retinas. Akt inhibitors significantly reduced retinal protein synthesis but did not affect glycolytic lactate production. Surprisingly, the glycolytic inhibitor 2-deoxyglucose (2-DG) markedly inhibited Akt1 and Akt3 activities, as well as protein synthesis. The effects of 2-DG, and 2-fluorodeoxyglucose (2-FDG) on retinal protein synthesis correlated with inhibition of lactate production and diminished ATP content, with all these effects reversed by provision of d-mannose. 2-DG treatment was not associated with increased AMPK, eEF2, or eIF2α phosphorylation; instead, it caused rapid dephosphorylation of 4E-BP1. 2-DG reduced total mTOR activity by 25%, but surprisingly, it did not reduce mTORC1 activity, as indicated by unaltered raptor-associated mTOR autophosphorylation and ribosomal protein S6 phosphorylation. Dephosphorylation of 4E-BP1 was largely prevented by inhibition of PP1/PP2A phosphatases with okadaic acid and calyculin A, and inhibition of PPM1 phosphatases with cadmium. Thus, inhibition of retinal glycolysis diminished Akt and protein synthesis coinciding with accelerated dephosphorylation of 4E-BP1 independently of mTORC1. These results demonstrate a novel mechanism regulating protein synthesis in the retina involving an mTORC1-independent and phosphatase-dependent regulation of 4E-BP1.

  5. The regulation of superoxide generation and nitric oxide synthesis by C-reactive protein.

    PubMed Central

    Ratnam, S; Mookerjea, S

    1998-01-01

    Activated macrophages utilize both reactive oxygen intermediates and reactive oxynitrogen intermediates for defence against microbes. However, simultaneous generation of superoxide (O- 2;) and nitric oxide (NO) could be harmful to host cells due to the production of peroxynitrite, nitrogen dioxide and hydroxyl radicals. Therefore, the regulation of the production of these molecules is critical to host survival. During periods of inflammation or infection, the level of serum C-reactive protein (CRP) increases in many species. Human and rat CRP have been shown to bind and interact with phagocytic cells. Since many of the interactions of CRP involve the binding to the phosphocholine ligand, we studied the role of CRP in O- 2; and NO generation through the modulation of phosphatidylcholine (PC) metabolism in macrophages. This study has shown that, while rat CRP inhibited phorbol myristate acetate- (PMA) induced release of O- 2; by rat macrophages, CRP-treated macrophages released NO in a time- and dose-dependent manner. CRP increased inducible nitric oxide synthase (iNOS) enzyme as well as iNOS mRNA levels in rat macrophages. Tricyclodecan-9-yl-xanthogenate (D609), an inhibitor to PC phospholipase C (PC-PLC), suppressed iNOS induction but enhanced PMA-induced release of O- 2;. These data indicate that an increased level of CRP during periods of inflammation may result in differential regulation of macrophage NADPH oxidase and iNOS activity. Increased hepatic synthesis of CRP may contribute to the mechanism by which phagocytic cells avoid simultaneous O- 2; and NO synthesis, and this could possibly be mediated through the regulation of PC-PLC. Images Figure 4 Figure 5 PMID:9767445

  6. Regulation of alpha-1 acid glycoprotein synthesis by porcine hepatocytes in monolayer culture.

    PubMed

    Caperna, T J; Shannon, A E; Stoll, M; Blomberg, L A; Ramsay, T G

    2015-07-01

    Alpha-1 acid glycoprotein (AGP, orosomucoid, ORM-1) is a highly glycosylated mammalian acute-phase protein, which is synthesized primarily in the liver and represents the major serum protein in newborn pigs. Recent data have suggested that the pig is unique in that AGP is a negative acute-phase protein in this species, and its circulating concentration appears to be associated with growth rate. The purpose of the present study was to investigate the regulation of AGP synthesis in hepatocytes prepared from suckling piglets and to provide a framework to compare its regulation with that of haptoglobin (HP), a positive acute-phase protein. Hepatocytes were isolated from preweaned piglets and maintained in serum-free monolayer culture for up to 72 h. The influences of hormones, cytokines, and redox modifiers on the expression and secretion of AGP and HP were determined by relative polymerase chain reaction and by measuring the concentration of each protein secreted into culture medium. The messenger RNA abundance and/or secretion of AGP protein was enhanced by interleukin (IL)-17a, IL-1, and resveratrol and inhibited by tumor necrosis factor-α (TNF), oncostatin M, and thyroid hormone (P < 0.05). HP expression and synthesis were upregulated by oncostatin M, IL-6, and dexamethasone and downregulated by TNF (P < 0.01). The overall messenger RNA expression at 24 h was in agreement with the secreted protein patterns confirming that control of these proteins in hepatocytes is largely transcriptional. Moreover, these data support the consideration that AGP is a negative acute-phase reactant and appears to be regulated by cytokines (with the exception of TNF) and hormones primarily in a manner opposite to that of the positive acute-phase protein, HP.

  7. Regulation of Viral RNA Synthesis by the V Protein of Parainfluenza Virus 5

    PubMed Central

    Yang, Yang; Zengel, James; Sun, Minghao; Sleeman, Katrina; Timani, Khalid Amine; Aligo, Jason; Rota, Paul

    2015-01-01

    ABSTRACT Paramyxoviruses include many important animal and human pathogens. The genome of parainfluenza virus 5 (PIV5), a prototypical paramyxovirus, encodes a V protein that inhibits viral RNA synthesis. In this work, the mechanism of inhibition was investigated. Using mutational analysis and a minigenome system, we identified regions in the N and C termini of the V protein that inhibit viral RNA synthesis: one at the very N terminus of V and the second at the C terminus of V. Furthermore, we determined that residues L16 and I17 are critical for the inhibitory function of the N-terminal region of the V protein. Both regions interact with the nucleocapsid protein (NP), an essential component of the viral RNA genome complex (RNP). Mutations at L16 and I17 abolished the interaction between NP and the N-terminal domain of V. This suggests that the interaction between NP and the N-terminal domain plays a critical role in V inhibition of viral RNA synthesis by the N-terminal domain. Both the N- and C-terminal regions inhibited viral RNA replication. The C terminus inhibited viral RNA transcription, while the N-terminal domain enhanced viral RNA transcription, suggesting that the two domains affect viral RNA through different mechanisms. Interestingly, V also inhibited the synthesis of the RNA of other paramyxoviruses, such as Nipah virus (NiV), human parainfluenza virus 3 (HPIV3), measles virus (MeV), mumps virus (MuV), and respiratory syncytial virus (RSV). This suggests that a common host factor may be involved in the replication of these paramyxoviruses. IMPORTANCE We identified two regions of the V protein that interact with NP and determined that one of these regions enhances viral RNA transcription via its interaction with NP. Our data suggest that a common host factor may be involved in the regulation of paramyxovirus replication and could be a target for broad antiviral drug development. Understanding the regulation of paramyxovirus replication will enable the

  8. Ceramide synthase-dependent ceramide generation and programmed cell death: involvement of salvage pathway in regulating postmitochondrial events.

    PubMed

    Mullen, Thomas D; Jenkins, Russell W; Clarke, Christopher J; Bielawski, Jacek; Hannun, Yusuf A; Obeid, Lina M

    2011-05-06

    The sphingolipid ceramide has been widely implicated in the regulation of programmed cell death or apoptosis. The accumulation of ceramide has been demonstrated in a wide variety of experimental models of apoptosis and in response to a myriad of stimuli and cellular stresses. However, the detailed mechanisms of its generation and regulatory role during apoptosis are poorly understood. We sought to determine the regulation and roles of ceramide production in a model of ultraviolet light-C (UV-C)-induced programmed cell death. We found that UV-C irradiation induces the accumulation of multiple sphingolipid species including ceramide, dihydroceramide, sphingomyelin, and hexosylceramide. Late ceramide generation was also found to be regulated by Bcl-xL, Bak, and caspases. Surprisingly, inhibition of de novo synthesis using myriocin or fumonisin B1 resulted in decreased overall cellular ceramide levels basally and in response to UV-C, but only fumonisin B1 inhibited cell death, suggesting the presence of a ceramide synthase (CerS)-dependent, sphingosine-derived pool of ceramide in regulating programmed cell death. We found that this pool did not regulate the mitochondrial pathway, but it did partially regulate activation of caspase-7 and, more importantly, was necessary for late plasma membrane permeabilization. Attempting to identify the CerS responsible for this effect, we found that combined knockdown of CerS5 and CerS6 was able to decrease long-chain ceramide accumulation and plasma membrane permeabilization. These data identify a novel role for CerS and the sphingosine salvage pathway in regulating membrane permeability in the execution phase of programmed cell death.

  9. RpiR Homologues May Link Staphylococcus aureus RNAIII Synthesis and Pentose Phosphate Pathway Regulation ▿ †

    PubMed Central

    Zhu, Yefei; Nandakumar, Renu; Sadykov, Marat R.; Madayiputhiya, Nandakumar; Luong, Thanh T.; Gaupp, Rosmarie; Lee, Chia Y.; Somerville, Greg A.

    2011-01-01

    Staphylococcus aureus is a medically important pathogen that synthesizes a wide range of virulence determinants. The synthesis of many staphylococcal virulence determinants is regulated in part by stress-induced changes in the activity of the tricarboxylic acid (TCA) cycle. One metabolic change associated with TCA cycle stress is an increased concentration of ribose, leading us to hypothesize that a pentose phosphate pathway (PPP)-responsive regulator mediates some of the TCA cycle-dependent regulatory effects. Using bioinformatics, we identified three potential ribose-responsive regulators that belong to the RpiR family of transcriptional regulators. To determine whether these RpiR homologues affect PPP activity and virulence determinant synthesis, the rpiR homologues were inactivated, and the effects on PPP activity and virulence factor synthesis were assessed. Two of the three homologues (RpiRB and RpiRC) positively influence the transcription of the PPP genes rpiA and zwf, while the third homologue (RpiRA) is slightly antagonistic to the other homologues. In addition, inactivation of RpiRC altered the temporal transcription of RNAIII, the effector molecule of the agr quorum-sensing system. These data confirm the close linkage of central metabolism and virulence determinant synthesis, and they establish a metabolic override for quorum-sensing-dependent regulation of RNAIII transcription. PMID:21926234

  10. Regulation of translesion DNA synthesis: Posttranslational modification of lysine residues in key proteins.

    PubMed

    McIntyre, Justyna; Woodgate, Roger

    2015-05-01

    Posttranslational modification of proteins often controls various aspects of their cellular function. Indeed, over the past decade or so, it has been discovered that posttranslational modification of lysine residues plays a major role in regulating translesion DNA synthesis (TLS) and perhaps the most appreciated lysine modification is that of ubiquitination. Much of the recent interest in ubiquitination stems from the fact that proliferating cell nuclear antigen (PCNA) was previously shown to be specifically ubiquitinated at K164 and that such ubiquitination plays a key role in regulating TLS. In addition, TLS polymerases themselves are now known to be ubiquitinated. In the case of human polymerase η, ubiquitination at four lysine residues in its C-terminus appears to regulate its ability to interact with PCNA and modulate TLS. Within the past few years, advances in global proteomic research have revealed that many proteins involved in TLS are, in fact, subject to a previously underappreciated number of lysine modifications. In this review, we will summarize the known lysine modifications of several key proteins involved in TLS; PCNA and Y-family polymerases η, ι, κ and Rev1 and we will discuss the potential regulatory effects of such modification in controlling TLS in vivo.

  11. MYB115 and MYB134 transcription factors regulate proanthocyanidin synthesis and structure.

    PubMed

    James, Amy Midori; Ma, Dawei; Mellway, Robin D; Gesell, Andreas; Yoshida, Kazuko; Walker, Vincent; Tran, Lan T; Stewart, Don; Reichelt, Michael; Suvanto, Jussi; Salminen, Juha-Pekka; Gershenzon, Jonathan; Seguin, Armand; Constabel, C Peter

    2017-03-27

    The accumulation of proanthocyanidins is regulated by a complex of transcription factors composed of R2R3 MYB, basic helix-loop-helix (bHLH), and WD-40 proteins which activate the promoters of biosynthetic genes. In poplar, MYB134 is known to regulate proanthocyanidin biosynthesis by activating key flavonoid genes. Here we characterize a second MYB regulator of proanthocyanidins, MYB115. Transgenic poplar overexpressing MYB115 showed a high proanthocyanidin phenotype and reduced salicinoid accumulation, similar to the effects of MYB134 overexpression. Transcriptomic analysis of MYB115- and MYB134-overexpressing poplar plants identified a set of common upregulated genes encoding proanthocyanidin biosynthetic enzymes and several novel uncharacterized MYB transcriptional repressors. Transient expression experiments demonstrated the capacity of both MYB134 and MYB115 to activate flavonoid promoters, but only in the presence of a bHLH cofactor. Yeast two-hybrid experiments confirmed the direct interaction of these transcription factors. The unexpected identification of dihydromyricetin in leaf extracts of both MYB115- and MYB134-overexpressing poplars led to the discovery of enhanced flavonoid B-ring hydroxylation and increased proportion of prodelphinidins in proanthocyanidin of the transgenics. The dramatic hydroxylation phenotype of MYB115 overexpressors is likely due to upregulation of both flavonoid 3,'5'- hydroxylases and cytochrome b5. Overall, this work provides new insight into the complexity of the gene regulatory network for proanthocyanidin synthesis in poplar.

  12. An Unconventional Diacylglycerol Kinase That Regulates Phospholipid Synthesis and Nuclear Membrane Growth*♦

    PubMed Central

    Han, Gil-Soo; O'Hara, Laura; Carman, George M.; Siniossoglou, Symeon

    2008-01-01

    Changes in nuclear size and shape during the cell cycle or during development require coordinated nuclear membrane remodeling, but the underlying molecular events are largely unknown. We have shown previously that the activity of the conserved phosphatidate phosphatase Pah1p/Smp2p regulates nuclear structure in yeast by controlling phospholipid synthesis and membrane biogenesis at the nuclear envelope. Two screens for novel regulators of phosphatidate led to the identification of DGK1. We show that Dgk1p is a unique diacylglycerol kinase that uses CTP, instead of ATP, to generate phosphatidate. DGK1 counteracts the activity of PAH1 at the nuclear envelope by controlling phosphatidate levels. Overexpression of DGK1 causes the appearance of phosphatidate-enriched membranes around the nucleus and leads to its expansion, without proliferating the cortical endoplasmic reticulum membrane. Mutations that decrease phosphatidate levels decrease nuclear membrane growth in pah1Δ cells. We propose that phosphatidate metabolism is a critical factor determining nuclear structure by regulating nuclear membrane biogenesis. PMID:18458075

  13. Disruption of sphingolipid biosynthesis in hepatocyte nodules: selective proliferative stimulus induced by fumonisin B1.

    PubMed

    van der Westhuizen, Liana; Gelderblom, Wentzel C A; Shephard, Gordon S; Swanevelder, Sonja

    2004-07-15

    In order to investigate the role of sphingolipid disruption in the cancer promoting potential of fumonisin B(1) (FB(1)) in the development of hepatocyte nodules, male Fischer 344 rats were subjected to cancer initiation (FB(1) containing diet or diethylnitrosamine (DEN) by i.p. injection) and promotion (2-acetylaminofluorene with partial hepatectomy, 2-AAF/PH) treatments followed by a secondary FB(1) dietary regimen. Sphinganine (Sa) and sphingosine (So) levels were measured by high performance liquid chromatography in control, surrounding and nodular liver tissues of the rats. The disruption of sphingolipid biosynthesis by the secondary FB(1) treatment in the control rats was significantly (P < 0.05) enhanced by the 2-AAF/PH cancer promotion treatment. The nodular and surrounding Sa levels returned to baseline following FB(1) initiation and 2-AAF/PH promotion. When comparing the groups subjected to the secondary FB(1) treatment, the initiation effected by FB(1) was less (P < 0.01) sensitive to the accumulation of Sa in the nodular and surrounding tissues than DEN initiation and the 2-AAF/PH control treatment. In contrast, the So level of FB(1) initiation was marginally increased in the nodules compared to the surrounding liver after 2-AAF/PH promotion and significantly (P < 0.05) higher with the secondary FB(1) treatment. Although, the FB(1)-induced hepatocyte nodules were not resistant to the disruption of sphingolipid biosynthesis, the nodular So levels were increased and might provide a selective growth stimulus possibly induced by bio-active sphingoid intermediates such as sphingosine 1-phosphate (S1P).

  14. Cell cycle specific distribution of killin: evidence for negative regulation of both DNA and RNA synthesis.

    PubMed

    Qiao, Man; Luo, Dan; Kuang, Yi; Feng, Haiyan; Luo, Guangping; Liang, Peng

    2015-01-01

    p53 tumor-suppressor gene is a master transcription factor which controls cell cycle progression and apoptosis. killin was discovered as one of the p53 target genes implicated in S-phase control coupled to cell death. Due to its extreme proximity to pten tumor-suppressor gene on human chromosome 10, changes in epigenetic modification of killin have also been linked to Cowden syndrome as well as other human cancers. Previous studies revealed that Killin is a high-affinity DNA-binding protein with preference to single-stranded DNA, and it inhibits DNA synthesis in vitro and in vivo. Here, co-localization studies of RFP-Killin with either GFP-PCNA or endogenous single-stranded DNA binding protein RPA during S-phase show that Killin always adopts a mutually exclusive punctuated nuclear expression pattern with the 2 accessory proteins in DNA replication. In contrast, when cells are not in S-phase, RFP-Killin largely congregates in the nucleolus where rRNA transcription normally occurs. Both of these cell cycle specific localization patterns of RFP-Killin are stable under high salt condition, consistent with Killin being tightly associated with nucleic acids within cell nuclei. Together, these cell biological results provide a molecular basis for Killin in competitively inhibiting the formation of DNA replication forks during S-phase, as well as potentially negatively regulate RNA synthesis during other cell cycle phases.

  15. Sugar-mediated semidian oscillation of gene expression in the cassava storage root regulates starch synthesis

    SciTech Connect

    Jansson, Christer; Baguma, Yona; Sun, Chuanxin; Boren, Mats; Olsson, Helena; Rosenqvist, Sara; Mutisya, Joel; Rubaihayo, Patrick R.; Jansson, Christer

    2008-01-15

    Starch branching enzyme (SBE) activity in the cassava storage root exhibited a diurnal fluctuation, dictated by a transcriptional oscillation of the corresponding SBE genes. The peak of SBE activity coincided with the onset of sucrose accumulation in the storage, and we conclude that the oscillatory mechanism keeps the starch synthetic apparatus in the storage root sink in tune with the flux of sucrose from the photosynthetic source. When storage roots were uncoupled from the source, SBE expression could be effectively induced by exogenous sucrose. Turanose, a sucrose isomer that cannot be metabolized by plants, mimicked the effect of sucrose, demonstrating that downstream metabolism of sucrose was not necessary for signal transmission. Also glucose and glucose-1-P induced SBE expression. Interestingly, induction by sucrose, turanose and glucose but not glucose-1-P sustained an overt semidian (12-h) oscillation in SBE expression and was sensitive to the hexokinase (HXK) inhibitor glucosamine. These results suggest a pivotal regulatory role for HXK during starch synthesis. Abscisic acid (ABA) was another potent inducer of SBE expression. Induction by ABA was similar to that of glucose-1-P in that it bypassed the semidian oscillator. Both the sugar and ABA signaling cascades were disrupted by okadaic acid, a protein phosphatase inhibitor. Based on these findings, we propose a model for sugar signaling in regulation of starch synthesis in the cassava storage root.

  16. Increased synthesis of folate transporters regulates folate transport in conditions of ethanol exposure and folate deficiency.

    PubMed

    Thakur, Shilpa; More, Deepti; Rahat, Beenish; Khanduja, Krishan Lal; Kaur, Jyotdeep

    2016-01-01

    Excessive alcohol consumption and dietary folate inadequacy are the main contributors leading to folate deficiency (FD). The present study was planned to study regulation of folate transport in conditions of FD and ethanol exposure in human embryonic kidney cell line. Also, the reversible nature of effects mediated by ethanol exposure and FD was determined by folate repletion and ethanol removal. For ethanol treatment, HEK293 cells were grown in medium containing 100 mM ethanol, and after treatment, one group of cells was shifted on medium that was free from ethanol. For FD treatment, cells were grown in folate-deficient medium followed by shifting of one group of cells on folate containing medium. FD as well as ethanol exposure resulted in an increase in folate uptake which was due to an increase in expression of folate transporters, i.e., reduced folate carrier, proton-coupled folate transporter, and folate receptor, both at the mRNA and protein level. The effects mediated by ethanol exposure and FD were reversible on removal of treatment. Promoter region methylation of folate transporters remained unaffected after FD and ethanol exposure. As far as transcription rate of folate transporters is concerned, an increase in rate of synthesis was observed in both ethanol exposure and FD conditions. Additionally, mRNA life of folate transporters was observed to be reduced by FD. An increased expression of folate transporters under ethanol exposure and FD conditions can be attributed to enhanced rate of synthesis of folate transporters.

  17. Small molecule modulators of eukaryotic initiation factor 2α kinases, the key regulators of protein synthesis.

    PubMed

    Joshi, Manali; Kulkarni, Abhijeet; Pal, Jayanta K

    2013-11-01

    Eukaryotic initiation factor 2 alpha kinases (eIF-2α kinases) are key mediators of stress response in cells. In mammalian cells, there are four eIF-2α kinases, namely HRI (Heme-Regulated Inhibitor), PKR (RNA-dependent Protein Kinase), PERK (PKR-like ER Kinase) and GCN2 (General Control Non-derepressible 2). These kinases get activated during diverse cytoplasmic stress conditions and phosphorylate the alpha-subunit of eIF2, leading to global protein synthesis inhibition. Therefore, eIF-2α kinases play a vital role in various cellular processes such as proliferation, differentiation, apoptosis and cell signaling. Deregulation of eIF-2α kinases and protein synthesis has been linked to numerous pathological conditions such as certain cancers, anemia and neurodegenerative disorders. Thus, modulation of these kinases by small molecules holds a great therapeutic promise. In this review we have compiled the available information on inhibitors and activators of these four eIF-2α kinases. The review concludes with a note on the selectivity issue of currently available modulators and future perspectives for the design of specific small molecule probes.

  18. Evaluation of Sphingolipids in Wistar Rats Treated to Prolonged and Single Oral Doses of Fumonisin B1

    PubMed Central

    Direito, Glória M.; Almeida, Adriana P.; Aquino, Simone; dos Reis, Tatiana Alves; Pozzi, Claudia Rodrigues; Corrêa, Benedito

    2009-01-01

    The objective of the present study was to evaluate sphingolipid levels (sphingosine-So and sphinganine-Sa) and to compare the Sa/So ratio in liver, serum and urine of Wistar rats after prolonged administration (21 days) of fumonisin B1 (FB1). In parallel, the kinetics of sphingolipid elimination in urine was studied in animals receiving a single dose of FB1. Prolonged exposure to FB1 caused an increase in Sa levels in urine, serum and liver. The most marked effect on sphingolipid biosynthesis was observed in animals treated with the highest dose of FB1. Animals receiving a single dose of FB1 presented variations in Sa and So levels and in the Sa/So ratio. PMID:19333435

  19. Royal jelly reduces melanin synthesis through down-regulation of tyrosinase expression.

    PubMed

    Han, Sang Mi; Yeo, Joo Hong; Cho, Yoon Hee; Pak, Sok Cheon

    2011-01-01

    For cosmetic reasons, the demand for effective and safe skin-whitening agents is high. Since the key enzyme in the melanin synthetic pathway is tyrosinase, many depigmenting agents in the treatment of hyperpigmentation act as tyrosinase inhibitors. In this study, we have investigated the hypo-pigmentary mechanism of royal jelly in a mouse melanocyte cell line, B16F1. Treatment of B16F1 cells with royal jelly markedly inhibited melanin biosynthesis in a dose-dependent manner. Decreased melanin content occurred through the decrease of tyrosinase activity. The mRNA levels of tyrosinase were also reduced by royal jelly. These results suggest that royal jelly reduces melanin synthesis by down-regulation of tyrosinase mRNA transcription and serves as a new candidate in the design of new skin-whitening or therapeutic agents.

  20. Translational regulation of protein synthesis, in response to light, at a critical stage of Volvox development

    SciTech Connect

    Kirk, M.M.; Kirk, D.L.

    1985-06-01

    In Volvox cultures synchronized by a light-dark cycle, juveniles containing presumptive somatic and reproductive cells are produced during the dark, but their cells do not differentiate until after the lights come on. The pattern of protein synthesis changes rapidly after the lights come on. Action spectra and effects of photosynthesis inhibitors indicate that this protein synthetic change is not simply a consequence of renewed flow of energy from illuminated chloroplasts. Actinomycin, at a level adequate to block the response to heat shock, has virtually no effect on the response of the same cells to light; furthermore, RNAs isolated from unilluminated and illuminated juveniles yield indistinguishable in vitro translation products. The authors conclude, therefore, that this effect of light is exerted almost exclusively at the translational level, generating one of the most striking examples of translational regulation yet described.

  1. Emergence of robust growth laws from optimal regulation of ribosome synthesis.

    PubMed

    Scott, Matthew; Klumpp, Stefan; Mateescu, Eduard M; Hwa, Terence

    2014-08-22

    Bacteria must constantly adapt their growth to changes in nutrient availability; yet despite large-scale changes in protein expression associated with sensing, adaptation, and processing different environmental nutrients, simple growth laws connect the ribosome abundance and the growth rate. Here, we investigate the origin of these growth laws by analyzing the features of ribosomal regulation that coordinate proteome-wide expression changes with cell growth in a variety of nutrient conditions in the model organism Escherichia coli. We identify supply-driven feedforward activation of ribosomal protein synthesis as the key regulatory motif maximizing amino acid flux, and autonomously guiding a cell to achieve optimal growth in different environments. The growth laws emerge naturally from the robust regulatory strategy underlying growth rate control, irrespective of the details of the molecular implementation. The study highlights the interplay between phenomenological modeling and molecular mechanisms in uncovering fundamental operating constraints, with implications for endogenous and synthetic design of microorganisms.

  2. Emergence of robust growth laws from optimal regulation of ribosome synthesis

    PubMed Central

    Scott, Matthew; Klumpp, Stefan; Mateescu, Eduard M; Hwa, Terence

    2014-01-01

    Bacteria must constantly adapt their growth to changes in nutrient availability; yet despite large-scale changes in protein expression associated with sensing, adaptation, and processing different environmental nutrients, simple growth laws connect the ribosome abundance and the growth rate. Here, we investigate the origin of these growth laws by analyzing the features of ribosomal regulation that coordinate proteome-wide expression changes with cell growth in a variety of nutrient conditions in the model organism Escherichia coli. We identify supply-driven feedforward activation of ribosomal protein synthesis as the key regulatory motif maximizing amino acid flux, and autonomously guiding a cell to achieve optimal growth in different environments. The growth laws emerge naturally from the robust regulatory strategy underlying growth rate control, irrespective of the details of the molecular implementation. The study highlights the interplay between phenomenological modeling and molecular mechanisms in uncovering fundamental operating constraints, with implications for endogenous and synthetic design of microorganisms. PMID:25149558

  3. Transfer RNA-mediated regulation of ribosome dynamics during protein synthesis.

    PubMed

    Fei, Jingyi; Richard, Arianne C; Bronson, Jonathan E; Gonzalez, Ruben L

    2011-08-21

    Translocation of tRNAs through the ribosome during protein synthesis involves large-scale structural rearrangement of the ribosome and ribosome-bound tRNAs that is accompanied by extensive and dynamic remodeling of tRNA-ribosome interactions. How the rearrangement of individual tRNA-ribosome interactions influences tRNA movement during translocation, however, remains largely unknown. To address this question, we used single-molecule FRET to characterize the dynamics of ribosomal pretranslocation (PRE) complex analogs carrying either wild-type or systematically mutagenized tRNAs. Our data reveal how specific tRNA-ribosome interactions regulate the rate of PRE complex rearrangement into a critical, on-pathway translocation intermediate and how these interactions control the stability of the resulting configuration. Notably, our results suggest that the conformational flexibility of the tRNA molecule has a crucial role in directing the structural dynamics of the PRE complex during translocation.

  4. Loss of Nuclear Receptor SHP Impairs but Does Not Eliminate Negative Feedback Regulation of Bile Acid Synthesis

    PubMed Central

    Kerr, Thomas A.; Saeki, Shigeru; Schneider, Manfred; Schaefer, Karen; Berdy, Sara; Redder, Thadd; Shan, Bei; Russell, David W.; Schwarz, Margrit

    2014-01-01

    Summary The in vivo role of the nuclear receptor SHP in feedback regulation of bile acid synthesis was examined. Loss of SHP in mice caused abnormal accumulation and increased synthesis of bile acids due to derepression of rate-limiting CYP7A1 and CYP8B1 hydroxylase enzymes in the biosynthetic pathway. Dietary bile acids induced liver damage and restored feedback regulation. A synthetic agonist of the nuclear receptor FXR was not hepatotoxic and had no regulatory effects. Reduction of the bile acid pool with cholestyramine enhanced CYP7A1 and CYP8B1 expression. We conclude that input from three negative regulatory pathways controls bile acid synthesis. One is mediated by SHP, and two are SHP independent and invoked by liver damage and changes in bile acid pool size. PMID:12062084

  5. Cyclic phosphatidic acid and lysophosphatidic acid induce hyaluronic acid synthesis via CREB transcription factor regulation in human skin fibroblasts.

    PubMed

    Maeda-Sano, Katsura; Gotoh, Mari; Morohoshi, Toshiro; Someya, Takao; Murofushi, Hiromu; Murakami-Murofushi, Kimiko

    2014-09-01

    Cyclic phosphatidic acid (cPA) is a naturally occurring phospholipid mediator and an analog of the growth factor-like phospholipid lysophosphatidic acid (LPA). cPA has a unique cyclic phosphate ring at the sn-2 and sn-3 positions of its glycerol backbone. We showed before that a metabolically stabilized cPA derivative, 2-carba-cPA, relieved osteoarthritis pathogenesis in vivo and induced hyaluronic acid synthesis in human osteoarthritis synoviocytes in vitro. This study focused on hyaluronic acid synthesis in human fibroblasts, which retain moisture and maintain health in the dermis. We investigated the effects of cPA and LPA on hyaluronic acid synthesis in human fibroblasts (NB1RGB cells). Using particle exclusion and enzyme-linked immunosorbent assays, we found that both cPA and LPA dose-dependently induced hyaluronic acid synthesis. We revealed that the expression of hyaluronan synthase 2 messenger RNA and protein is up-regulated by cPA and LPA treatment time dependently. We then characterized the signaling pathways up-regulating hyaluronic acid synthesis mediated by cPA and LPA in NB1RGB cells. Pharmacological inhibition and reporter gene assays revealed that the activation of the LPA receptor LPAR1, Gi/o protein, phosphatidylinositol-3 kinase (PI3K), extracellular-signal-regulated kinase (ERK), and cyclic adenosine monophosphate response element-binding protein (CREB) but not nuclear factor κB induced hyaluronic acid synthesis by the treatment with cPA and LPA in NB1RGB cells. These results demonstrate for the first time that cPA and LPA induce hyaluronic acid synthesis in human skin fibroblasts mainly through the activation of LPAR1-Gi/o followed by the PI3K, ERK, and CREB signaling pathway.

  6. Sphingolipid transfer proteins defined by the GLTP-fold

    PubMed Central

    Malinina, Lucy; Simanshu, Dhirendra K.; Zhai, Xiuhong; Samygina, Valeria R.; Kamlekar, RaviKanth; Kenoth, Roopa; Ochoa-Lizarralde, Borja; Malakhova, Margarita L.; Molotkovsky, Julian G.; Patel, Dinshaw J.; Brown, Rhoderick E.

    2015-01-01

    Glycolipid transfer proteins (GLTPs) originally were identified as small (~24 kDa), soluble, amphitropic proteins that specifically accelerate the intermembrane transfer of glycolipids. GLTPs and related homologs now are known to adopt a unique, helically dominated, two-layer ‘sandwich’ architecture defined as the GLTP-fold that provides the structural underpinning for the eukaryotic GLTP superfamily. Recent advances now provide exquisite insights into structural features responsible for lipid headgroup selectivity as well as the adaptability of the hydrophobic compartment for accommodating hydrocarbon chains of differing length and unsaturation. A new understanding of the structural versatility and evolutionary premium placed on the GLTP motif has emerged. Human GLTP-motifs have evolved to function not only as glucosylceramide binding/transferring domains for phosphoinositol 4-phosphate adaptor protein-2 during glycosphingolipid biosynthesis but also as selective binding/transfer proteins for ceramide-1-phosphate. The latter, known as ceramide-l-phosphate transfer protein, recently has been shown to form GLTP-fold while critically regulating Group-IV cytoplasmic phospholipase A2 activity and pro-inflammatory eicosanoid production. PMID:25797198

  7. Up-regulation of tryptophan hydroxylase expression and serotonin synthesis by sertraline.

    PubMed

    Kim, Seong Who; Park, So Yeon; Hwang, Onyou

    2002-04-01

    The neurotransmitter serotonin is involved in a variety of brain functions, and abnormal changes in serotonin neurotransmission are associated with an array of psychiatric disorders, including depression. Sertraline is a selective serotonin reuptake inhibitor (SSRI) and an effective antidepressant. Sertraline increases the serotonin concentration in the synaptic cleft by a short-term action; however, clinical improvement is observed only after several weeks, suggesting that the therapeutic effect may be caused by long-term alterations in serotonin transmission. We determined the effects of sertraline on serotonin synthesis in vivo and in vitro. Long-term treatment of rats with sertraline up-regulated mRNA and protein levels of the serotonin-synthesizing enzyme tryptophan hydroxylase (TPH), as determined by in situ hybridization and immunocytochemistry, respectively. In vitro studies using RBL-2H3 cells also showed an increase in mRNA and protein levels of TPH by sertraline, as determined by Northern blot and immunoblot analyses, respectively. This was accompanied by increases in the levels of TPH enzymatic activity and total serotonin. These data demonstrate that in addition to the known short-term action as an uptake blocker, sertraline also exerts a long-term effect on the serotonin neurotransmission by enhancing serotonin synthesis. A similar effect was observed with another SSRI, fluoxetine, but not with the non-SSRI chlorpromazine. The up-regulation of TPH gene expression by sertraline was attenuated by the protein kinase A (PKA) inhibitor N-[2-(p-bromocinnamylamine)-ethyl]-5-isoquinolinesulfonamine, suggesting that a mechanism involving the PKA signaling pathway might at least in part mediate the long-term therapeutic action.

  8. The insulin/TOR signal transduction pathway is involved in the nutritional regulation of juvenile hormone synthesis in Aedes aegypti.

    PubMed

    Pérez-Hedo, Meritxell; Rivera-Perez, Crisalejandra; Noriega, Fernando G

    2013-06-01

    Juvenile hormone (JH) levels must be modulated to permit the normal progress of development and reproductive maturation in mosquitoes. JH is part of a transduction system that assesses nutritional information and controls reproduction in mosquitoes. Adult female Aedes aegypti show nutritionally-dependent dynamic changes in corpora allata (CA) JH biosynthetic activities. A coordinated expression of most JH biosynthetic enzymes has been described in female pupae and adult mosquitoes; increases or decreases in transcript levels for all the enzymes were concurrent with increases or decreases in JH synthesis; suggesting that transcriptional changes are at least partially responsible for the dynamic changes of JH biosynthesis. The goal of the present study is to identify signaling network components responsible for the nutritional-dependent changes of JH synthesis in the CA of mosquitoes. The insulin/TOR signaling network plays a central role in the transduction of nutritional signals that regulate cell growth and metabolism in insects. These pathways have also been suggested as a link between nutritional signals and JH synthesis regulation in the CA of cockroaches and flies. We used a combination of in vitro studies and in vivo genetic knockdown experiments to explore nutritional signaling pathways in the CA. Our results suggest that the insulin/TOR pathway plays a role in the transduction of the nutritional information that regulates JH synthesis in mosquitoes. Transcriptional regulation of the genes encoding JH biosynthetic enzymes is at least partially responsible for these nutritionally modulated changes of JH biosynthesis.

  9. Oxygen-dependent regulation of c-di-GMP synthesis by SadC controls alginate production in Pseudomonas aeruginosa.

    PubMed

    Schmidt, Annika; Hammerbacher, Anna Silke; Bastian, Mike; Nieken, Karen Jule; Klockgether, Jens; Merighi, Massimo; Lapouge, Karine; Poschgan, Claudia; Kölle, Julia; Acharya, K Ravi; Ulrich, Martina; Tümmler, Burkhard; Unden, Gottfried; Kaever, Volkhard; Lory, Stephen; Haas, Dieter; Schwarz, Sandra; Döring, Gerd

    2016-10-01

    Pseudomonas aeruginosa produces increased levels of alginate in response to oxygen-deprived conditions. The regulatory pathway(s) that links oxygen limitation to increased synthesis of alginate has remained elusive. In the present study, using immunofluorescence microscopy, we show that anaerobiosis-induced alginate production by planktonic PAO1 requires the diguanylate cyclase (DGC) SadC, previously identified as a regulator of surface-associated lifestyles. Furthermore, we found that the gene products of PA4330 and PA4331, located in a predicted operon with sadC, have a major impact on alginate production: deletion of PA4330 (odaA, for oxygen-dependent alginate synthesis activator) caused an alginate production defect under anaerobic conditions, whereas a PA4331 (odaI, for oxygen-dependent alginate synthesis inhibitor) deletion mutant produced alginate also in the presence of oxygen, which would normally inhibit alginate synthesis. Based on their sequence, OdaA and OdaI have predicted hydratase and dioxygenase reductase activities, respectively. Enzymatic assays using purified protein showed that unlike OdaA, which did not significantly affect DGC activity of SadC, OdaI inhibited c-di-GMP production by SadC. Our data indicate that SadC, OdaA and OdaI are components of a novel response pathway of P. aeruginosa that regulates alginate synthesis in an oxygen-dependent manner.

  10. Nutritional Signaling Regulates Vitellogenin Synthesis and Egg Development through Juvenile Hormone in Nilaparvata lugens (Stål)

    PubMed Central

    Lu, Kai; Chen, Xia; Liu, Wen-Ting; Zhang, Xin-Yu; Chen, Ming-Xiao; Zhou, Qiang

    2016-01-01

    Insect female reproduction which comprises the synthesis of vitellogenein (Vg) in the fat body and its incorporation into developing oocytes, needs a large amount of energy and food resources. Our previous studies found that juvenile hormone (JH) regulates vitellogenesis in the brown planthopper, Nilaparvata lugens. Here, we report on the role of JH in nutrient-regulated Vg synthesis and egg development. We first cloned the genes coding for juvenile hormone acid methyltransferase (JHAMT) which is involved in JH biosynthesis and methoprene-tolerant (Met) for JH action. Amino acids (AAs) induced the expression of jmtN, while showing no effects on the expression of met using an artificial diet culture system. Reduction in JH biosynthesis or its action by RNA interference (RNAi)-mediated silencing of jmtN or met led to a severe inhibition of AAs-induced Vg synthesis and oocyte maturation, together with lower fecundity. Furthermore, exogenous application of JH III partially restored Vg expression levels in jmtN RNAi females. However, JH III application did not rescue Vg synthesis in these met RNAi insects. Our results show that AAs induce Vg synthesis in the fat body and egg development in concert with JH biosynthesis in Nilaparvata lugens (Stål), rather than through JH action. PMID:26927076

  11. Nutritional Signaling Regulates Vitellogenin Synthesis and Egg Development through Juvenile Hormone in Nilaparvata lugens (Stål).

    PubMed

    Lu, Kai; Chen, Xia; Liu, Wen-Ting; Zhang, Xin-Yu; Chen, Ming-Xiao; Zhou, Qiang

    2016-02-26

    Insect female reproduction which comprises the synthesis of vitellogenein (Vg) in the fat body and its incorporation into developing oocytes, needs a large amount of energy and food resources. Our previous studies found that juvenile hormone (JH) regulates vitellogenesis in the brown planthopper, Nilaparvata lugens. Here, we report on the role of JH in nutrient-regulated Vg synthesis and egg development. We first cloned the genes coding for juvenile hormone acid methyltransferase (JHAMT) which is involved in JH biosynthesis and methoprene-tolerant (Met) for JH action. Amino acids (AAs) induced the expression of jmtN, while showing no effects on the expression of met using an artificial diet culture system. Reduction in JH biosynthesis or its action by RNA interference (RNAi)-mediated silencing of jmtN or met led to a severe inhibition of AAs-induced Vg synthesis and oocyte maturation, together with lower fecundity. Furthermore, exogenous application of JH III partially restored Vg expression levels in jmtN RNAi females. However, JH III application did not rescue Vg synthesis in these met RNAi insects. Our results show that AAs induce Vg synthesis in the fat body and egg development in concert with JH biosynthesis in Nilaparvata lugens (Stål), rather than through JH action.

  12. Regulation of the synthesis of pulp degrading enzymes in Bacillus isolated from cocoa fermentation.

    PubMed

    Ouattara, Honoré G; Reverchon, Sylvie; Niamke, Sébastien L; Nasser, William

    2017-05-01

    Pectin degrading enzymes are essential for quality of product from cocoa fermentation. Previously, we studied purified pectate lyases (Pel) produced by Bacillus strains from fermenting cocoa and characterized the cloned pel genes. This study aims to search for biological signals that modulates Pel production and regulators that influence pel gene expression. Strains were grown to the end of exponential phase in media containing various carbon sources. Pel enzymes production in Bacillus was unaffected by simple sugar content variation up to 2%. Additionally, it appeared that pel gene is not under the control of the most common carbon and pectin catabolism regulators ccpA and kdgR, which could explain the insensitivity of Pel production to carbon source variation. However, a 6-fold decrease in Pel production was observed when bacteria were grown in LB rich medium as opposed to basal mineral medium. Subsequently, bioinformatics analysis of cloned pel gene promoter region revealed the presence of DegU binding site. Furthermore, the deletion of degU gene dramatically reduces the pel gene expression, as revealed by real time quantitative PCR, showing an activation effect of DegU on Pel synthesis in Bacillus strains studied. We assumed that, during the latter stage of cocoa fermentation when simple sugars are depleted, production of Pel in Bacillus is stimulated by DegU to supply microbial cells with carbon source from polymeric pectic compounds.

  13. GAD67-mediated GABA Synthesis and Signaling Regulate Inhibitory Synaptic Innervation in the Visual Cortex

    PubMed Central

    Chattopadhyaya, Bidisha; Di Cristo, Graziella; Wu, Cai Zhi; Knott, Graham; Kuhlman, Sandra; Fu, Yu; Palmiter, Richard D.; Huang, Z. Josh

    2007-01-01

    The development of GABAergic inhibitory circuits is shaped by neural activity, but the underlying mechanisms are unclear. we demonstrate a novel function of GABA in regulating GABAergic innervation in the adolescent brain, when GABA is mainly known as an inhibitory transmitter. Conditional knockdown of the rate-limiting synthetic enzyme GAD67 in basket interneurons in adolescent visual cortex resulted in cell autonomous deficits in axon branching, perisomatic synapse formation around pyramidal neurons, and complexity of the innervation fields; the same manipulation had little influence on the subsequent maintenance of perisomatic synapses. These effects of GABA deficiency were rescued by suppressing GABA re-uptake and by GABA receptor agonists. Germ-line knockdown of GAD67 but not GAD65 showed similar deficits, suggesting a specific role of GAD67 in the maturation of perisomatic innervation. Since intracellular GABA levels are modulated by neuronal activity, our results implicate GAD67-mediated GABA synthesis in activity-dependent regulation of inhibitory innervation patterns. PMID:17582330

  14. ESCRT-II controls retinal axon growth by regulating DCC receptor levels and local protein synthesis.

    PubMed

    Konopacki, Filip A; Wong, Hovy Ho-Wai; Dwivedy, Asha; Bellon, Anaïs; Blower, Michael D; Holt, Christine E

    2016-04-01

    Endocytosis and local protein synthesis (LPS) act coordinately to mediate the chemotropic responses of axons, but the link between these two processes is poorly understood. The endosomal sorting complex required for transport (ESCRT) is a key regulator of cargo sorting in the endocytic pathway, and here we have investigated the role of ESCRT-II, a critical ESCRT component, in Xenopus retinal ganglion cell (RGC) axons. We show that ESCRT-II is present in RGC axonal growth cones (GCs) where it co-localizes with endocytic vesicle GTPases and, unexpectedly, with the Netrin-1 receptor, deleted in colorectal cancer (DCC). ESCRT-II knockdown (KD) decreases endocytosis and, strikingly, reduces DCC in GCs and leads to axon growth and guidance defects. ESCRT-II-depleted axons fail to turn in response to a Netrin-1 gradient in vitro and many axons fail to exit the eye in vivo These defects, similar to Netrin-1/DCC loss-of-function phenotypes, can be rescued in whole (in vitro) or in part (in vivo) by expressing DCC. In addition, ESCRT-II KD impairs LPS in GCs and live imaging reveals that ESCRT-II transports mRNAs in axons. Collectively, our results show that the ESCRT-II-mediated endocytic pathway regulates both DCC and LPS in the axonal compartment and suggest that ESCRT-II aids gradient sensing in GCs by coupling endocytosis to LPS.

  15. Cloning and transcriptional regulation of genes responsible for synthesis of gangliosides.

    PubMed

    Zeng, Guichao; Yu, Robert K

    2008-04-01

    Ganglioside synthases are glycosyltransferases involved in the biosynthesis of glycoconjugates. A number of ganglioside synthase genes have been cloned and characterized. They are classified into different families of glycosyltransferases based on similarities of their amino acid sequences. Tissue-specific expression of these genes has been analyzed by hybridization using cDNA fragments. Enzymatic characterization with the expressed recombinant enzymes showed these enzymes differ in their donor and acceptor substrate specificities and other biochemical parameters. In vitro enzymatic analysis also showed that one linkage can be synthesized by multiple enzymes and one enzyme may be responsible for synthesis of multiple gangliosides. Following the cloning of the ganglioside synthase genes, the promoters of the key synthase genes in the ganglioside biosynthetic pathway have been cloned and analyzed. All of the promoters are TATA-less, lacking a CCAAT box but containing GC-rich boxes, characteristic of the house-keeping genes, although transcription of ganglioside synthase genes is subject to complex developmental and tissue-specific regulation. A set of cis-acting elements and transcription factors, including Sp1, AP2, and CREB, function in the proximal promoters. Negative-regulatory regions have also been defined in most of the promoters. We present here an overview of these genes and their transcriptional regulation.

  16. Legionella pneumophila S1P-lyase targets host sphingolipid metabolism and restrains autophagy

    PubMed Central

    Rolando, Monica; Escoll, Pedro; Nora, Tamara; Botti, Joëlle; Boitez, Valérie; Daniels, Craig; Abraham, Gilu; Stogios, Peter J.; Skarina, Tatiana; Christophe, Charlotte; Dervins-Ravault, Delphine; Cazalet, Christel; Hilbi, Hubert; Rupasinghe, Thusitha W. T.; Tull, Dedreia; McConville, Malcolm J.; Ong, Sze Ying; Hartland, Elizabeth L.; Codogno, Patrice; Levade, Thierry; Naderer, Thomas; Savchenko, Alexei; Buchrieser, Carmen

    2016-01-01

    Autophagy is an essential component of innate immunity, enabling the detection and elimination of intracellular pathogens. Legionella pneumophila, an intracellular pathogen that can cause a severe pneumonia in humans, is able to modulate autophagy through the action of effector proteins that are translocated into the host cell by the pathogen’s Dot/Icm type IV secretion system. Many of these effectors share structural and sequence similarity with eukaryotic proteins. Indeed, phylogenetic analyses have indicated their acquisition by horizontal gene transfer from a eukaryotic host. Here we report that L. pneumophila translocates the effector protein sphingosine-1 phosphate lyase (LpSpl) to target the host sphingosine biosynthesis and to curtail autophagy. Our structural characterization of LpSpl and its comparison with human SPL reveals high structural conservation, thus supporting prior phylogenetic analysis. We show that LpSpl possesses S1P lyase activity that was abrogated by mutation of the catalytic site residues. L. pneumophila triggers the reduction of several sphingolipids critical for macrophage function in an LpSpl-dependent and -independent manner. LpSpl activity alone was sufficient to prevent an increase in sphingosine levels in infected host cells and to inhibit autophagy during macrophage infection. LpSpl was required for efficient infection of A/J mice, highlighting an important virulence role for this effector. Thus, we have uncovered a previously unidentified mechanism used by intracellular pathogens to inhibit autophagy, namely the disruption of host sphingolipid biosynthesis. PMID:26831115

  17. Modulation of sphingolipid metabolism with calorie restriction enhances insulin action in skeletal muscle

    PubMed Central

    Obanda, Diana N.; Yu, Yongmei; Wang, Zhong Q; Cefalu, William T.

    2015-01-01

    This study sought to investigate the effect of calorie restriction (CR) on skeletal muscle sphingolipid metabolism and its contribution to improved insulin action in rats after a 6 month feeding study. Twenty nine (29) male Fischer-344 rats were randomized to an ad libitum (AL) diet or 30% CR. Dietary intake, body weight, and insulin sensitivity were monitored. After 6 months, skeletal muscle (vastus lateralis) was obtained for insulin signaling and lipid profiling. Calorie restriction significantly decreased insulin and glucose levels and also altered the expression and activity of proteins involved in sphingolipid formation and metabolism. The quantities of ceramides significantly increased in CR animals (p<0.05; n=14–15), while ceramide metabolism products (i.e glycosphingolipids: hexosylceramides and lactosylceramides) significantly decreased (p<0.05; n=14–15). Ceramide phosphates, sphingomyelins, sphingosine and sphingosine phosphate were not significantly different between AL and CR groups (p=ns; n=14–15). Lactosylceramide quantities correlated significantly with surrogate markers of insulin resistance (HOMA-IR) (r=0.7, p<0.005). Products of ceramide metabolism (glycosphingolipids), known to interfere with insulin signaling at elevated levels were significantly reduced in the skeletal muscle of CR animals. The increase in insulin sensitivity is associated with glycosphingolipid levels. PMID:25771159

  18. Legionella pneumophila S1P-lyase targets host sphingolipid metabolism and restrains autophagy.

    PubMed

    Rolando, Monica; Escoll, Pedro; Nora, Tamara; Botti, Joëlle; Boitez, Valérie; Bedia, Carmen; Daniels, Craig; Abraham, Gilu; Stogios, Peter J; Skarina, Tatiana; Christophe, Charlotte; Dervins-Ravault, Delphine; Cazalet, Christel; Hilbi, Hubert; Rupasinghe, Thusitha W T; Tull, Dedreia; McConville, Malcolm J; Ong, Sze Ying; Hartland, Elizabeth L; Codogno, Patrice; Levade, Thierry; Naderer, Thomas; Savchenko, Alexei; Buchrieser, Carmen

    2016-02-16

    Autophagy is an essential component of innate immunity, enabling the detection and elimination of intracellular pathogens. Legionella pneumophila, an intracellular pathogen that can cause a severe pneumonia in humans, is able to modulate autophagy through the action of effector proteins that are translocated into the host cell by the pathogen's Dot/Icm type IV secretion system. Many of these effectors share structural and sequence similarity with eukaryotic proteins. Indeed, phylogenetic analyses have indicated their acquisition by horizontal gene transfer from a eukaryotic host. Here we report that L. pneumophila translocates the effector protein sphingosine-1 phosphate lyase (LpSpl) to target the host sphingosine biosynthesis and to curtail autophagy. Our structural characterization of LpSpl and its comparison with human SPL reveals high structural conservation, thus supporting prior phylogenetic analysis. We show that LpSpl possesses S1P lyase activity that was abrogated by mutation of the catalytic site residues. L. pneumophila triggers the reduction of several sphingolipids critical for macrophage function in an LpSpl-dependent and -independent manner. LpSpl activity alone was sufficient to prevent an increase in sphingosine levels in infected host cells and to inhibit autophagy during macrophage infection. LpSpl was required for efficient infection of A/J mice, highlighting an important virulence role for this effector. Thus, we have uncovered a previously unidentified mechanism used by intracellular pathogens to inhibit autophagy, namely the disruption of host sphingolipid biosynthesis.

  19. A procedure for fractionation of sphingolipid classes by solid-phase extraction on aminopropyl cartridges.

    PubMed

    Bodennec, J; Koul, O; Aguado, I; Brichon, G; Zwingelstein, G; Portoukalian, J

    2000-09-01

    Solid-phase extraction (SPE) methods are easy, rapid, and reliable. Their growing popularity is in part due to their operational simplicity and cost reduction in solvents, and partly because they are easier to automate. Sphingolipids are implicated in various cellular events such as growth, differentiation, and apoptosis. However, their separation by small SPE cartridges has attracted limited attention. Here we describe an SPE procedure on aminopropyl cartridges that by sequential elution allows the separation of a lipid mixture into free ceramides, neutral glycosphingolipids, neutral phospholipids (sphingomyelin), and a fraction containing the acidic phospholipids and phosphorylated sphingoid bases, phosphoceramides and sulfatides. Individual components are obtained in high yield and purity. We applied the procedure to obtain data on separation of [(3)H]myristic acid-labeled sphingolipids from fish gills, and from human melanoma tumor tissue. Individual lipids in the SPE fractions were identified by chromatography on several high-performance thin-layer chromatography (HPTLC) systems. The chromatographic behavior of free sphingoid bases is also reported.

  20. Functional identification of a delta8-sphingolipid desaturase from Borago officinalis.

    PubMed

    Sperling, P; Libisch, B; Zähringer, U; Napier, J A; Heinz, E

    2001-04-15

    The similarities between delta12- and delta5-fatty acyl desaturase sequences were used to construct degenerate primers for PCR experiments with cDNA transcribed from mRNA of developing borage seeds. Screening of a borage seed cDNA library with an amplified DNA fragment resulted in the isolation of a full-length cDNA corresponding to a deduced open-reading frame of 446 amino acids. The protein showed high similarity to plant delta8-sphingolipid desaturases as well as to the delta6-fatty acyl desaturase from Borago officinalis. The sequence is characterized by the presence of a N-terminal cytochrome b5 domain. Expression of this open-reading frame in Saccharomyces cerevisiae resulted in the formation of delta8-trans/cis-phytosphingenines not present in wild-type cells, as shown by HPLC analysis of sphingoid bases as their dinitrophenyl derivatives. GLC-MS analysis of the methylated di-O-trimethylsilyl ether derivatives confirmed the presence of delta8-stereoisomers of C18- and C20-phytosphingenine. Furthermore, Northern blotting showed that the gene encoding a stereo-unselective delta8-sphingolipid desaturase is primarily expressed in young borage leaves.

  1. Enzymological mechanism for the regulation of lanthanum chloride on flavonoid synthesis of soybean seedlings under enhanced ultraviolet-B radiation.

    PubMed

    Fan, Caixia; Hu, Huiqing; Wang, Lihong; Zhou, Qing; Huang, Xiaohua

    2014-01-01

    In order to probe into the enzymological mechanism for the regulation of lanthanum chloride (LaCl3) on flavonoid synthesis in plants under enhanced ultraviolet-B (UV-B) radiation, the effects of LaCl₃ (20 and 60 mg l(-1)) on the content of flavonoids as well as the activities of phenylalanine ammonia-lyase (PAL), cinnamate-4-hydroxylase (C4H), 4-coumarate : coenzyme A ligase (4CL), and chalcone synthase (CHS) in soybean seedlings under enhanced UV-B radiation (2.6 and 6.2 kJ m(-2) day(-1)) were investigated. Enhanced UV-B radiation (2.6 and 6.2 kJ m(-2) day(-1)) caused the increase in the content of flavonoids as well as the activities of PAL, C4H, 4CL, and CHS in soybean seedlings. The treatment of 20 mg l(-1) LaCl₃ also efficiently increased these indices, which promoted the flavonoid synthesis and provided protective effects for resisting enhanced UV-B radiation. On the contrary, the treatment of 60 mg l(-1) LaCl₃ decreased the content of flavonoids as well as the activities of C4H, 4CL, and CHS in soybean seedlings except increasing the activity of PAL, which were not beneficial to the flavonoid synthesis and provided negative effects for resisting enhanced UV-B radiation. In conclusion, enhanced UV-B radiation caused the increase in the flavonoid synthesis by promoting the activities of PAL, C4H, 4CL, and CHS in soybean seedlings. The treatment of LaCl₃ could change flavonoid synthesis in soybean seedlings under enhanced UV-B radiation by regulating the activities of PAL, C4H, 4CL, and CHS, which is an enzymological mechanism for the regulation of LaCl₃ on flavonoid synthesis in plants under enhanced UV-B radiation.

  2. Monocyte/macrophage-derived microparticles up-regulate inflammatory mediator synthesis by human airway epithelial cells.

    PubMed

    Cerri, Chiara; Chimenti, Daniele; Conti, Ilaria; Neri, Tommaso; Paggiaro, Pierluigi; Celi, Alessandro

    2006-08-01

    Cell-derived microparticles (MP) are membrane fragments shed by virtually all eukaryotic cells upon activation or during apoptosis that play a significant role in physiologically relevant processes, including coagulation and inflammation. We investigated whether MP derived from monocytes/macrophages have the potential to modulate human airway epithelial cell activation. Monocytes/macrophages were isolated from the buffy coats of blood donors by Ficoll gradient centrifugation, followed by overnight culture of the mononuclear cell fraction. Adherent cells were washed and incubated with the calcium ionophore, A23187, or with histamine. The MP-containing supernatant was incubated with cells of the human bronchial epithelial line BEAS-2B and of the human alveolar line A549. IL-8, MCP-1, and ICAM-1 production was assessed by ELISA and by RT-PCR. In some experiments, monocytes/macrophages were stained with the fluorescent lipid intercalating dye PKH67, and the supernatant was analyzed by FACS. Stimulation of monocytes/macrophages with A23187 caused the release of particles that retain their fluorescent lipid intercalating label, indicating that they are derived from cell membranes. Incubation with A549 and BEAS-2B cells up-regulate IL-8 synthesis. Ultrafiltration and ultracentrifugation of the material abolished the effect, indicating that particulate matter, rather than soluble molecules, is responsible for it. Up-regulation of MCP-1 and ICAM-1 was also demonstrated in A549 cells. Similar results were obtained with histamine. Our data show that human monocytes/macrophages release MP that have the potential to sustain the innate immunity of the airway epithelium, as well as to contribute to the pathogenesis of inflammatory diseases of the lungs through up-regulation of proinflammatory mediators.

  3. Regulation of cholesterol synthesis in cultured mouse mammary carcinoma FM3A cells.

    PubMed

    Hasumi, K; Otsuki, R; Endo, A

    1985-08-01

    Mouse mammary carcinoma FM3A cells, which are able to grow in a serum-free medium, have novel characteristics that could be valuable in biochemical and somatic cell genetic studies. In FM3A cells grown in the presence of serum, both sterol synthesis and the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the major rate-limiting enzyme in the cholesterol biosynthetic pathway, were strongly suppressed by human low density lipoprotein (LDL). The addition of LDL (50 micrograms protein/ml) resulted in a 50% decrease in the reductase activity within 3 h and a 95% reduction after 24 h. Similarly, over 90% suppression of the reductase activity was obtained by the addition of LDL or mevalonolactone when the cells were grown on a serum-free medium. ML-236B (compactin), a specific inhibitor of HMG-CoA reductase, inhibited sterol synthesis from [14C]acetate by 80% at 1 microM. Reductase activity in FM3A cells was increased by 2.5- to 5-fold when the cells were treated with ML-236B (at 0.26-2.6 microM for 24 h). Thus, in FM3A cells, HMG-CoA reductase activity responded well to LDL, as is observed in human skin fibroblasts. Along with other novel features of this cell line, the present observations indicate that FM3A cells should be useful in biochemical and somatic cell genetic analysis of cholesterol metabolism, especially as regards the regulation of HMG-CoA reductase activity.

  4. Differential regulation of membrane and secretory mu chain synthesis in human beta cell lines. Regulation of membrane mu or secreted mu

    PubMed Central

    1982-01-01

    Regulation of membrane and secretory mu synthesis was examined in human lymphoblastoid cell lines representing various stages of differentiation. Immunoglobulin phenotype was determined by surface and cytoplasmic staining with fluorochrome-conjugated antibodies and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of anti-mu precipitable cellular products. The thymidine analogue, 5-bromo-2'-deoxyuridine (BUdR), which inhibits differentiation-specific proteins in a variety of systems, was used to examine regulation of immunoglobulin synthesis. We found that BUdR had a differential effect on membrane (mum) and secretory (mus) type mu heavy chains. Ig production in pre-B and plasma cell-like lines, which make mus, was unaffected by BUdR. However, surface expression of IgM (mum) in B cell lines was drastically inhibited at similar doses of BUdR without diminishing total Ig or protein synthesis. Examination of labeled mu chains from control and BUdR-treated B cell lines by SDS- PAGE revealed the production of two sizes of mu (mum and mus) in control cells and only the smaller size (mus) in BUdR-treated cells. This size difference could not be attributed to alterations in glycosylation of the molecules. These data show that BUdR inhibits the production of membrane mu chains without diminishing secretory mu chain synthesis in the same cell. Our findings suggest that thymidine-rich regions of the genome are involved in the regulation of mum vs. mus during B cell differentiation. PMID:6816895

  5. CYTOCHROME P450 REGULATION: THE INTERPLAY BETWEEN ITS HEME AND APOPROTEIN MOIETIES IN SYNTHESIS, ASSEMBLY, REPAIR AND DISPOSAL123

    PubMed Central

    Correia, Maria Almira; Sinclair, Peter R.; De Matteis, Francesco

    2011-01-01

    Heme is vital to our aerobic universe. Heme cellular content is finely tuned through an exquisite control of synthesis and degradation. Heme deficiency is deleterious to cells, whereas excess heme is toxic. Most of the cellular heme serves as the prosthetic moiety of functionally diverse hemoproteins, including cytochromes P450 (P450s). In the liver, P450s are its major consumers with >50% of hepatic heme committed to their synthesis. Prosthetic heme is the sine qua non of P450 catalytic biotransformation of both endo- and xenobiotics. This well-recognized functional role notwithstanding, heme also regulates P450 protein synthesis, assembly, repair and disposal. These less well-appreciated aspects are reviewed herein. PMID:20860521

  6. MicroRNAs in the pineal gland: miR-483 regulates melatonin synthesis by targeting arylalkylamine N-acetyltransferase.

    PubMed

    Clokie, Samuel J H; Lau, Pierre; Kim, Hyun Hee; Coon, Steven L; Klein, David C

    2012-07-20

    MicroRNAs (miRNAs) play a broad range of roles in biological regulation. In this study, rat pineal miRNAs were profiled for the first time, and their importance was evaluated by focusing on the main function of the pineal gland, melatonin synthesis. Massively parallel sequencing and related methods revealed the miRNA population is dominated by a small group of miRNAs as follows: ~75% is accounted for by 15 miRNAs; miR-182 represents 28%. In addition to miR-182, miR-183 and miR-96 are also highly enriched in the pineal gland, a distinctive pattern also found in the retina. This effort also identified previously unrecognized miRNAs and other small noncoding RNAs. Pineal miRNAs do not exhibit a marked night/day difference in abundance with few exceptions (e.g. 2-fold night/day differences in the abundance of miR-96 and miR-182); this contrasts sharply with the dynamic 24-h pattern that characterizes the pineal transcriptome. During development, the abundance of most pineal gland-enriched miRNAs increases; however, there is a marked decrease in at least one, miR-483. miR-483 is a likely regulator of melatonin synthesis, based on the following. It inhibits melatonin synthesis by pinealocytes in culture; it acts via predicted binding sites in the 3"-UTR of arylalkylamine N-acetyltransferase (Aanat) mRNA, the penultimate enzyme in melatonin synthesis, and it exhibits a developmental profile opposite to that of Aanat transcripts. Additionally, a miR-483 targeted antagonist increased melatonin synthesis in neonatal pinealocytes. These observations support the hypothesis that miR-483 suppresses Aanat mRNA levels during development and that the developmental decrease in miR-483 abundance promotes melatonin synthesis.

  7. mTOR complex 1 signalling regulates the balance between lipid synthesis and oxidation in hypoxia lymphocytes

    PubMed Central

    Yin, Geng; Liang, Yan; Wang, Ying; Yang, Yuan; Yang, Min; Cen, Xiao-min

    2017-01-01

    Mammalian cells adapt to different environmental conditions and alter cellular metabolic pathways to meet the energy demand for survival. Thus, the metabolic regulation of cells under special conditions, such as hypoxia, should be precisely regulated. During the metabolic regulation, mammalian target of rapamycin (mTOR) plays a vital role in the sensing of extracellular stimulations and regulating intracellular adaptations. Here, we report that mTOR complex 1 (mTORC1) signalling is a central regulator of lipid homoeostasis in lymphocytes. In hypoxia, mTORC1 activity is reduced and shifts lipid synthesis to lipid oxidation. Moreover, knockdown tuberous sclerosis complex 1 (TSC1) constitutively activates mTORC1 activity and impairs the hypoxia-induced metabolic shift. Therefore, TSC1 knockdown enhances hypoxia-induced cell death. Re-inactivation of mTORC1 activity via rapamycin may resist hypoxia-induced cell death in TSC1 knockdown lymphocytes. Our findings provide a deep insight into mTORC1 in the metabolic balance of lipid synthesis and oxidation, and imply that mTORC1 activity should be precisely regulated for the lipid homoeostasis in lymphocytes. PMID:28057888

  8. mTOR complex 1 signalling regulates the balance between lipid synthesis and oxidation in hypoxia lymphocytes.

    PubMed

    Yin, Geng; Liang, Yan; Wang, Ying; Yang, Yuan; Yang, Min; Cen, Xiao-Min; Xie, Qi-Bing

    2017-02-28

    Mammalian cells adapt to different environmental conditions and alter cellular metabolic pathways to meet the energy demand for survival. Thus, the metabolic regulation of cells under special conditions, such as hypoxia, should be precisely regulated. During the metabolic regulation, mammalian target of rapamycin (mTOR) plays a vital role in the sensing of extracellular stimulations and regulating intracellular adaptations. Here, we report that mTOR complex 1 (mTORC1) signalling is a central regulator of lipid homoeostasis in lymphocytes. In hypoxia, mTORC1 activity is reduced and shifts lipid synthesis to lipid oxidation. Moreover, knockdown tuberous sclerosis complex 1 (TSC1) constitutively activates mTORC1 activity and impairs the hypoxia-induced metabolic shift. Therefore, TSC1 knockdown enhances hypoxia-induced cell death. Re-inactivation of mTORC1 activity via rapamycin may resist hypoxia-induced cell death in TSC1 knockdown lymphocytes. Our findings provide a deep insight into mTORC1 in the metabolic balance of lipid synthesis and oxidation, and imply that mTORC1 activity should be precisely regulated for the lipid homoeostasis in lymphocytes.

  9. UAP56 is a novel interacting partner of Bcr in regulating vascular smooth muscle cell DNA synthesis

    SciTech Connect

    Sahni, Abha; Wang, Nadan; Alexis, Jeffrey D.

    2012-04-13

    Highlights: Black-Right-Pointing-Pointer UAP56 is an important regulator of DNA synthesis in vascular smooth muscle cells. Black-Right-Pointing-Pointer UAP56 binds to Bcr. Black-Right-Pointing-Pointer Interaction between Bcr and UAP56 is critical for Bcr induced DNA synthesis. -- Abstract: Bcr is a serine/threonine kinase that is a critical regulator of vascular smooth muscle cell inflammation and proliferation. We have previously demonstrated that Bcr acts in part via phosphorylation and inhibition of PPAR{gamma}. We have identified the RNA helicase UAP56 as another substrate of Bcr. In this report we demonstrate that knockdown of UAP56 blocks Bcr induced DNA synthesis in vascular smooth muscle cells (VSMC). We also found that over expression of Bcr increased the expression of cyclin E and decreased the expression of p27. Knockdown of UAP56 reversed the effect of Bcr on cyclin E and p27 expression. Furthermore, we found that Bcr binds to UAP56 and demonstrate that binding of UAP56 to Bcr is critical for Bcr induced DNA synthesis in VSMC. Our data identify UAP56 as an important binding partner of Bcr and a novel target for inhibiting vascular smooth muscle cell proliferation.

  10. IL-15 regulates memory CD8+ T cell O-glycan synthesis and affects trafficking

    PubMed Central

    Nolz, Jeffrey C.; Harty, John T.

    2014-01-01

    Memory and naive CD8+ T cells exhibit distinct trafficking patterns. Specifically, memory but not naive CD8+ T cells are recruited to inflamed tissues in an antigen-independent manner. However, the molecular mechanisms that regulate memory CD8+ T cell trafficking are largely unknown. Here, using murine models of infection and T cell transfer, we found that memory but not naive CD8+ T cells dynamically regulate expression of core 2 O-glycans, which interact with P- and E-selectins to modulate trafficking to inflamed tissues. Following infection, antigen-specific effector CD8+ T cells strongly expressed core 2 O-glycans, but this glycosylation pattern was lost by most memory CD8+ T cells. After unrelated infection or inflammatory challenge, memory CD8+ T cells synthesized core 2 O-glycans independently of antigen restimulation. The presence of core 2 O-glycans subsequently directed these cells to inflamed tissue. Memory and naive CD8+ T cells exhibited the opposite pattern of epigenetic modifications at the Gcnt1 locus, which encodes the enzyme that initiates core 2 O-glycan synthesis. The open chromatin configuration in memory CD8+ T cells permitted de novo generation of core 2 O-glycans in a TCR-independent, but IL-15–dependent, manner. Thus, IL-15 stimulation promotes antigen-experienced memory CD8+ T cells to generate core 2 O-glycans, which subsequently localize them to inflamed tissues. These findings suggest that CD8+ memory T cell trafficking potentially can be manipulated to improve host defense and immunotherapy. PMID:24509081

  11. Regulation of lipid synthesis genes and milk fat production in human mammary epithelial cells during secretory activation.

    PubMed

    Mohammad, Mahmoud A; Haymond, Morey W

    2013-09-15

    Expression of genes for lipid biosynthetic enzymes during initiation of lactation in humans is unknown. Our goal was to study mRNA expression of lipid metabolic enzymes in human mammary epithelial cell (MEC) in conjunction with the measurement of milk fatty acid (FA) composition during secretory activation. Gene expression from mRNA isolated from milk fat globule (MFG) and milk FA composition were measured from 6 h to 42 days postpartum in seven normal women. Over the first 96 h postpartum, daily milk fat output increased severalfold and mirrored expression of genes for all aspects of lipid metabolism and milk FA production, including lipolysis at the MEC membrane, FA uptake from blood, intracellular FA transport, de novo FA synthesis, FA and glycerol activation, FA elongation, FA desaturation, triglyceride synthesis, cholesterol synthesis, and lipid droplet formation. Expression of the gene for a key lipid synthesis regulator, sterol regulatory element-binding transcription factor 1 (SREBF1), increased 2.0-fold by 36 h and remained elevated over the study duration. Expression of genes for estrogen receptor 1, thyroid hormone-responsive protein, and insulin-induced 2 increased progressively to plateau by 96 h. In contrast, mRNA of peroxisome proliferator-activated receptor-γ decreased severalfold. With onset of lactation, increased de novo synthesis of FA was the most prominent change in milk FA composition and mirrored the expression of FA synthesis genes. In conclusion, milk lipid synthesis and secretion in humans is a complex process requiring the orchestration of a wide variety of pathways of which SREBF1 may play a primary role.

  12. Phosphatidylcholine synthesis in the rat: The substrate for methylation and regulation by choline

    SciTech Connect

    Datko, A.H.; Aksamit, R.R.; Mudd, S.H. )

    1990-03-01

    Two lines of evidence led us to reexamine the possibility that methylation of phosphoethanolamine and its partially methylated derivatives, in addition to methylation of the corresponding phosphatidyl derivatives, plays a role in mammalian phosphatidylcholine biosynthesis: (a) Results obtained by Salerno and Beeler with rat appear to strongly support such a role for methylation of phosphobases; (b) Such reactions have recently been shown to play major roles in phosphatidylcholine synthesis by higher plants. We found that, following continuous labeling of rat liver with L-(methyl-3H)methionine for 10.4 min (intraperitoneal administration) or for 0.75 min (intraportal administration), virtually no 3H was detected in methylated derivatives of phosphoethanolamine, but readily detectable amounts of 3H were present in the base moiety of each methylated derivative of phosphatidylethanolamine. Thus, there was no indication that phospho-base methylation makes a significant contribution. Studies of cultured rat hepatoma cells showed definitively for the first time in a mammalian system that choline deprivation up-regulates the rate of flow of methyl groups originating in methionine into phosphatidylethanolamine and derivatives. Even under these conditions, methylation of phosphoethanolamine bases appeared to play a negligible role.

  13. Evidence for differential photic regulation of pineal melatonin synthesis in teleosts.

    PubMed

    Migaud, H; Davie, A; Martinez Chavez, C C; Al-Khamees, S

    2007-11-01

    The aim of this study was to compare the circadian control of melatonin production in teleosts. To do so, the effects of ophthalmectomy on circulating melatonin rhythms were studied along with ex vivo pineal culture in six different teleosts. Results strongly suggested that the circadian control of melatonin production could have dramatically changed with at least three different systems being present in teleosts when one considers the photic regulation of pineal melatonin production. First, salmonids presented a decentralized system in which the pineal gland responds directly to light independently of the eyes. Then, in seabass and cod both the eyes and the pineal gland are required to sustain full night-time melatonin production. Finally, a third type of circadian control of melatonin production is proposed in tilapia and catfish in which the pineal gland would not be light sensitive (or only slightly) and required the eyes to perceive light and inhibit melatonin synthesis. Further studies (anatomical, ultrastructural, retinal projections) are needed to confirm these results. Ex vivo experiments indirectly confirmed these results, as while the pineal gland responded normally to day-night rhythms in salmonids, seabass and cod, only very low levels were obtained at night in tilapia and no melatonin could be measured from isolated pineal glands in catfish. Together, these findings suggest that mechanisms involved in the perception of light and the transduction of this signal through the circadian axis has changed in teleosts possibly as a reflection of the photic environment in which they have evolved in.

  14. Death-associated Protein 3 Regulates Mitochondrial-encoded Protein Synthesis and Mitochondrial Dynamics.

    PubMed

    Xiao, Lin; Xian, Hongxu; Lee, Kit Yee; Xiao, Bin; Wang, Hongyan; Yu, Fengwei; Shen, Han-Ming; Liou, Yih-Cherng

    2015-10-09

    Mitochondrial morphologies change over time and are tightly regulated by dynamic machinery proteins such as dynamin-related protein 1 (Drp1), mitofusion 1/2, and optic atrophy 1 (OPA1). However, the detailed mechanisms of how these molecules cooperate to mediate fission and fusion remain elusive. DAP3 is a mitochondrial ribosomal protein that involves in apoptosis, but its biological function has not been well characterized. Here, we demonstrate that DAP3 specifically localizes in the mitochondrial matrix. Knockdown of DAP3 in mitochondria leads to defects in mitochondrial-encoded protein synthesis and abnormal mitochondrial dynamics. Moreover, depletion of DAP3 dramatically decreases the phosphorylation of Drp1 at Ser-637 on mitochondria, enhancing the retention time of Drp1 puncta on mitochondria during the fission process. Furthermore, autophagy is inhibited in the DAP3-depleted cells, which sensitizes cells to different types of death stimuli. Together, our results suggest that DAP3 plays important roles in mitochondrial function and dynamics, providing new insights into the mechanism of a mitochondrial ribosomal protein function in cell death.

  15. Nitric oxide synthesis in the lung. Regulation by oxygen through a kinetic mechanism.

    PubMed Central

    Dweik, R A; Laskowski, D; Abu-Soud, H M; Kaneko, F; Hutte, R; Stuehr, D J; Erzurum, S C

    1998-01-01

    In this study, we show that oxygen regulates nitric oxide (NO) levels through effects on NO synthase (NOS) enzyme kinetics. Initially, NO synthesis in the static lung was measured in bronchiolar gases during an expiratory breath-hold in normal individuals. NO accumulated exponentially to a plateau, indicating balance between NO production and consumption in the lung. Detection of NO2-, NO3-, and S-nitrosothiols in lung epithelial lining fluids confirmed NO consumption by chemical reactions in the lung. Interestingly, alveolar gas NO (estimated from bronchiolar gases at end-expiration) was near zero, suggesting NO in exhaled gases is not derived from circulatory/systemic sources. Dynamic NO levels during tidal breathing in different airway regions (mouth, trachea, bronchus, and bronchiole) were similar. However, in individuals breathing varying levels of inspired oxygen, dynamic NO levels were notably dependent on O2 concentration in the hypoxic range (KmO2 190 microM). Purified NOS type II enzyme activity in vitro was similarly dependent on molecular oxygen levels (KmO2 135 microM), revealing a means by which oxygen concentration affects NO levels in vivo. Based upon these results, we propose that NOS II is a mediator of the vascular response to oxygen in the lung, because its KmO2 allows generation of NO in proportion to the inspired oxygen concentration throughout the physiologic range. PMID:9449700

  16. Facile synthesis of diverse graphene nanomeshes based on simultaneous regulation of pore size and surface structure

    PubMed Central

    Zhang, Jia; Song, Huaibing; Zeng, Dawen; Wang, Hao; Qin, Ziyu; Xu, Keng; Pang, Aimin; Xie, Changsheng

    2016-01-01

    Recently, graphene nanomesh (GNM) has attracted great attentions due to its unique porous structure, abundant active sites, finite band gap and possesses potential applications in the fields of electronics, gas sensor/storage, catalysis, etc. Therefore, diverse GNMs with different physical and chemical properties are required urgently to meet different applications. Herein we demonstrate a facile synthetic method based on the famous Fenton reaction to prepare GNM, by using economically fabricated graphene oxide (GO) as a starting material. By precisely controlling the reaction time, simultaneous regulation of pore size from 2.9 to 11.1 nm and surface structure can be realized. Ultimately, diverse GNMs with tunable band gap and work function can be obtained. Specially, the band gap decreases from 4.5–2.3 eV for GO, which is an insulator, to 3.9–1.24 eV for GNM-5 h, which approaches to a semiconductor. The dual nature of electrophilic addition and oxidizability of HO• is responsible for this controllable synthesis. This efficient, low-cost, inherently scalable synthetic method is suitable for provide diverse and optional GNMs, and may be generalized to a universal technique. PMID:27561350

  17. Transfer RNA-mediated regulation of ribosome dynamics during protein synthesis

    PubMed Central

    Fei, Jingyi; Richard, Arianne C.; Bronson, Jonathan E.; Gonzalez, & Ruben L.

    2011-01-01

    Translocation of transfer RNAs (tRNAs) through the ribosome during protein synthesis involves large-scale structural rearrangements of the ribosome and the ribosome-bound tRNAs that are accompanied by extensive and dynamic remodeling of tRNA-ribosome interactions. The contributions that rearranging individual tRNA-ribosome interactions make to directing tRNA movements during translocation, however, remain largely unknown. To address this question, we have used single-molecule fluorescence resonance energy transfer to characterize the dynamics of ribosomal pre-translocation (PRE) complex analogs carrying either wild-type or systematically mutagenized tRNAs. Our data reveal how specific tRNA-ribosome interactions regulate the rate with which the PRE complex rearranges into a critical, on-pathway translocation intermediate and how these interactions control the stability of the resulting configuration. More interestingly, our results suggest that the conformational flexibility of the tRNA molecule itself plays a crucial role in directing the structural dynamics of the PRE complex during translocation. PMID:21857664

  18. Biglycan- and Sphingosine Kinase-1 Signaling Crosstalk Regulates the Synthesis of Macrophage Chemoattractants

    PubMed Central

    Hsieh, Louise Tzung-Harn; Nastase, Madalina-Viviana; Roedig, Heiko; Zeng-Brouwers, Jinyang; Poluzzi, Chiara; Schwalm, Stephanie; Fork, Christian; Tredup, Claudia; Brandes, Ralf P.; Wygrecka, Malgorzata; Huwiler, Andrea; Pfeilschifter, Josef; Schaefer, Liliana

    2017-01-01

    In its soluble form, the extracellular matrix proteoglycan biglycan triggers the synthesis of the macrophage chemoattractants, chemokine (C-C motif) ligand CCL2 and CCL5 through selective utilization of Toll-like receptors (TLRs) and their adaptor molecules. However, the respective downstream signaling events resulting in biglycan-induced CCL2 and CCL5 production have not yet been defined. Here, we show that biglycan stimulates the production and activation of sphingosine kinase 1 (SphK1) in a TLR4- and Toll/interleukin (IL)-1R domain-containing adaptor inducing interferon (IFN)-β (TRIF)-dependent manner in murine primary macrophages. We provide genetic and pharmacological proof that SphK1 is a crucial downstream mediator of biglycan-triggered CCL2 and CCL5 mRNA and protein expression. This is selectively driven by biglycan/SphK1-dependent phosphorylation of the nuclear factor NF-κB p65 subunit, extracellular signal-regulated kinase (Erk)1/2 and p38 mitogen-activated protein kinases. Importantly, in vivo overexpression of soluble biglycan causes Sphk1-dependent enhancement of renal CCL2 and CCL5 and macrophage recruitment into the kidney. Our findings describe the crosstalk between biglycan- and SphK1-driven extracellular matrix- and lipid-signaling. Thus, SphK1 may represent a new target for therapeutic intervention in biglycan-evoked inflammatory conditions. PMID:28282921

  19. Facile synthesis of diverse graphene nanomeshes based on simultaneous regulation of pore size and surface structure

    NASA Astrophysics Data System (ADS)

    Zhang, Jia; Song, Huaibing; Zeng, Dawen; Wang, Hao; Qin, Ziyu; Xu, Keng; Pang, Aimin; Xie, Changsheng

    2016-08-01

    Recently, graphene nanomesh (GNM) has attracted great attentions due to its unique porous structure, abundant active sites, finite band gap and possesses potential applications in the fields of electronics, gas sensor/storage, catalysis, etc. Therefore, diverse GNMs with different physical and chemical properties are required urgently to meet different applications. Herein we demonstrate a facile synthetic method based on the famous Fenton reaction to prepare GNM, by using economically fabricated graphene oxide (GO) as a starting material. By precisely controlling the reaction time, simultaneous regulation of pore size from 2.9 to 11.1 nm and surface structure can be realized. Ultimately, diverse GNMs with tunable band gap and work function can be obtained. Specially, the band gap decreases from 4.5–2.3 eV for GO, which is an insulator, to 3.9–1.24 eV for GNM-5 h, which approaches to a semiconductor. The dual nature of electrophilic addition and oxidizability of HO• is responsible for this controllable synthesis. This efficient, low-cost, inherently scalable synthetic method is suitable for provide diverse and optional GNMs, and may be generalized to a universal technique.

  20. Regulated Synthesis and Functions of Laminin 5 in Polarized Madin-Darby Canine Kidney Epithelial Cells

    PubMed Central

    Mak, Grace Z.; Kavanaugh, Gina M.; Buschmann, Mary M.; Stickley, Shaun M.; Koch, Manuel; Goss, Kathleen Heppner; Waechter, Holly; Zuk, Anna

    2006-01-01

    Renal tubular epithelial cells synthesize laminin (LN)5 during regeneration of the epithelium after ischemic injury. LN5 is a truncated laminin isoform of particular importance in the epidermis, but it is also constitutively expressed in a number of other epithelia. To investigate the role of LN5 in morphogenesis of a simple renal epithelium, we examined the synthesis and function of LN5 in the spreading, proliferation, wound-edge migration, and apical–basal polarization of Madin-Darby canine kidney (MDCK) cells. MDCK cells synthesize LN5 only when subconfluent, and they degrade the existing LN5 matrix when confluent. Through the use of small-interfering RNA to knockdown the LN5 α3 subunit, we were able to demonstrate that LN5 is necessary for cell proliferation and efficient wound-edge migration, but not apical–basal polarization. Surprisingly, suppression of LN5 production caused cells to spread much more extensively than normal on uncoated surfaces, and exogenous keratinocyte LN5 was unable to rescue this phenotype. MDCK cells also synthesized laminin α5, a component of LN10, that independent studies suggest may form an assembled basal lamina important for polarization. Overall, our findings indicate that LN5 is likely to play an important role in regulating cell spreading, migration, and proliferation during reconstitution of a continuous epithelium. PMID:16775009

  1. HOPS: a novel cAMP-dependent shuttling protein involved in protein synthesis regulation.

    PubMed

    Della Fazia, Maria Agnese; Castelli, Marilena; Bartoli, Daniela; Pieroni, Stefania; Pettirossi, Valentina; Piobbico, Danilo; Viola-Magni, Mariapia; Servillo, Giuseppe

    2005-07-15

    The liver has the ability to autonomously regulate growth and mass. Following partial hepatectomy, hormones, growth factors, cytokines and their coupled signal transduction pathways have been implicated in hepatocyte proliferation. To understand the mechanisms responsible for the proliferative response, we studied liver regeneration by characterization of novel genes that are activated in residual hepatocytes. A regenerating liver cDNA library screening was performed with cDNA-subtracted probes derived from regenerating and normal liver. Here, we describe the biology of Hops (for hepatocyte odd protein shuttling). HOPS is a novel shuttling protein that contains an ubiquitin-like domain, a putative NES and a proline-rich region. HOPS is rapidly exported from the nucleus and is overexpressed during liver regeneration. Evidence shows that cAMP governs HOPS export in hepatocytes of normal and regenerating liver and is mediated via CRM-1. We demonstrate that HOPS binds to elongation factor eEF-1A and interferes in protein synthesis. HOPS overexpression in H-35-hepatoma and 3T3-NIH cells strongly reduces proliferation.

  2. The Sphingolipid Biosynthetic Pathway Is a Potential Target for Chemotherapy against Chagas Disease

    PubMed Central

    Koeller, Carolina Macedo; Heise, Norton

    2011-01-01

    The protozoan parasite Trypanosoma cruzi is the causative agent of human Chagas disease, for which there currently is no cure. The life cycle of T. cruzi is complex, including an extracellular phase in the triatomine insect vector and an obligatory intracellular stage inside the vertebrate host. These phases depend on a variety of surface glycosylphosphatidylinositol-(GPI-) anchored glycoconjugates that are synthesized by the parasite. Therefore, the surface expression of GPI-anchored components and the biosynthetic pathways of GPI anchors are attractive targets for new therapies for Chagas disease. We identified new drug targets for chemotherapy by taking the available genome sequence information and searching for differences in the sphingolipid biosynthetic pathways (SBPs) of mammals and T. cruzi. In this paper, we discuss the major steps of the SBP in mammals, yeast and T. cruzi, focusing on the IPC synthase and ceramide remodeling of T. cruzi as potential therapeutic targets for Chagas disease. PMID:21603271

  3. Serum profiling of healthy aging identifies phospho- and sphingolipid species as markers of human longevity.

    PubMed

    Montoliu, Ivan; Scherer, Max; Beguelin, Fiona; DaSilva, Laeticia; Mari, Daniela; Salvioli, Stefano; Martin, Francois-Pierre J; Capri, Miriam; Bucci, Laura; Ostan, Rita; Garagnani, Paolo; Monti, Daniela; Biagi, Elena; Brigidi, Patrizia; Kussmann, Martin; Rezzi, Serge; Franceschi, Claudio; Collino, Sebastiano

    2014-01-01

    As centenarians well represent the model of healthy aging, there are many important implications in revealing the underlying molecular mechanisms behind such successful aging. By combining NMR metabonomics and shot-gun lipidomics in serum we analyzed metabolome and lipidome composition of a group of centenarians with respect to elderly individuals. Specifically, NMR metabonomics profiling of serum revealed that centenarians are characterized by a metabolic phenotype distinct from that of elderly subjects, in particular regarding amino acids and lipid species. Shot- gun lipidomics approach displays unique changes in lipids biosynthesis in centenarians, with 41 differently abundant lipid species with respect to elderly subjects. These findings reveal phospho/sphingolipids as putative markers and biological modulators of healthy aging, in humans. Considering the particular actions of these metabolites, these data are suggestive of a better counteractive antioxidant capacity and a well-developed membrane lipid remodelling process in the healthy aging phenotype.

  4. Nitric oxide-sphingolipid interplays in plant signalling: a new enigma from the Sphinx?

    PubMed

    Guillas, Isabelle; Puyaubert, Juliette; Baudouin, Emmanuel

    2013-09-12

    Nitric oxide (NO) emerged as one of the major signaling molecules operating during plant development and plant responses to its environment. Beyond the identification of the direct molecular targets of NO, a series of studies considered its interplay with other actors of signal transduction and the integration of NO into complex signaling networks. Beside the close relationships between NO and calcium or phosphatidic acid signaling pathways that are now well-established, recent reports paved the way for interplays between NO and sphingolipids (SLs). This mini-review summarizes our current knowledge of the influence NO and SLs might exert on each other in plant physiology. Based on comparisons with examples from the animal field, it further indicates that, although SL-NO interplays are common features in signaling networks of eukaryotic cells, the underlying mechanisms and molecular targets significantly differ.

  5. TOR Pathway-Mediated Juvenile Hormone Synthesis Regulates Nutrient-Dependent Female Reproduction in Nilaparvata lugens (Stål).

    PubMed

    Lu, Kai; Chen, Xia; Liu, Wen-Ting; Zhou, Qiang

    2016-03-28

    The "target of rapamycin" (TOR) nutritional signaling pathway and juvenile hormone (JH) regulation of vitellogenesis has been known for a long time. However, the interplay between these two pathways regulating vitellogenin (Vg) expression remains obscure. Here, we first demonstrated the key role of amino acids (AAs) in activation of Vg synthesis and egg development in Nilaparvata lugens using chemically defined artificial diets. AAs induced the expression of TOR and S6K (S6 kinase), whereas RNAi-mediated silencing of these two TOR pathway genes and rapamycin application strongly inhibited the AAs-induced Vg synthesis. Furthermore, knockdown of Rheb (Ras homologue enriched in brain), TOR, S6K and application of rapamycin resulted in a dramatic reduction in the mRNA levels of jmtN (juvenile hormone acid methyltransferase, JHAMT). Application of JH III on the RNAi (Rheb and TOR) and rapamycin-treated females partially rescued the Vg expression. Conversely, knockdown of either jmtN or met (methoprene-tolerant, JH receptor) and application of JH III had no effects on mRNA levels of Rheb, TOR and S6K and phosphorylation of S6K. In summary, our results demonstrate that the TOR pathway induces JH biosynthesis that in turn regulates AAs-mediated Vg synthesis in N. lugens.

  6. TOR Pathway-Mediated Juvenile Hormone Synthesis Regulates Nutrient-Dependent Female Reproduction in Nilaparvata lugens (Stål)

    PubMed Central

    Lu, Kai; Chen, Xia; Liu, Wen-Ting; Zhou, Qiang

    2016-01-01

    The “target of rapamycin” (TOR) nutritional signaling pathway and juvenile hormone (JH) regulation of vitellogenesis has been known for a long time. However, the interplay between these two pathways regulating vitellogenin (Vg) expression remains obscure. Here, we first demonstrated the key role of amino acids (AAs) in activation of Vg synthesis and egg development in Nilaparvata lugens using chemically defined artificial diets. AAs induced the expression of TOR and S6K (S6 kinase), whereas RNAi-mediated silencing of these two TOR pathway genes and rapamycin application strongly inhibited the AAs-induced Vg synthesis. Furthermore, knockdown of Rheb (Ras homologue enriched in brain), TOR, S6K and application of rapamycin resulted in a dramatic reduction in the mRNA levels of jmtN (juvenile hormone acid methyltransferase, JHAMT). Application of JH III on the RNAi (Rheb and TOR) and rapamycin-treated females partially rescued the Vg expression. Conversely, knockdown of either jmtN or met (methoprene-tolerant, JH receptor) and application of JH III had no effects on mRNA levels of Rheb, TOR and S6K and phosphorylation of S6K. In summary, our results demonstrate that the TOR pathway induces JH biosynthesis that in turn regulates AAs-mediated Vg synthesis in N. lugens. PMID:27043527

  7. The role of mTOR signaling in the regulation of protein synthesis and muscle mass during immobilization in mice

    PubMed Central

    You, Jae-Sung; Anderson, Garrett B.; Dooley, Matthew S.; Hornberger, Troy A.

    2015-01-01

    ABSTRACT The maintenance of skeletal muscle mass contributes substantially to health and to issues associated with the quality of life. It has been well recognized that skeletal muscle mass is regulated by mechanically induced changes in protein synthesis, and that signaling by mTOR is necessary for an increase in protein synthesis and the hypertrophy that occurs in response to increased mechanical loading. However, the role of mTOR signaling in the regulation of protein synthesis and muscle mass during decreased mechanical loading remains largely undefined. In order to define the role of mTOR signaling, we employed a mouse model of hindlimb immobilization along with pharmacological, mechanical and genetic means to modulate mTOR signaling. The results first showed that immobilization induced a decrease in the global rates of protein synthesis and muscle mass. Interestingly, immobilization also induced an increase in mTOR signaling, eIF4F complex formation and cap-dependent translation. Blocking mTOR signaling during immobilization with rapamycin not only impaired the increase in eIF4F complex formation, but also augmented the decreases in global protein synthesis and muscle mass. On the other hand, stimulating immobilized muscles with isometric contractions enhanced mTOR signaling and rescued the immobilization-induced decrease in global protein synthesis through a rapamycin-sensitive mechanism that was independent of ribosome biogenesis. Unexpectedly, the effects of isometric contractions were also independent of eIF4F complex formation. Similar to isometric contractions, overexpression of Rheb in immobilized muscles enhanced mTOR signaling, cap-dependent translation and global protein synthesis, and prevented the reduction in fiber size. Therefore, we conclude that the activation of mTOR signaling is both necessary and sufficient to alleviate the decreases in protein synthesis and muscle mass that occur during immobilization. Furthermore, these results indicate

  8. Spingolipids in the root play an important role in regulating the leaf ionome in Arabidopsis thaliana

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sphingolipid synthesis is initiated by condensation of serine with palmitoyl-CoA to produce 3-ketodihydrosphinganine (3-KDS), which is subsequently reduced by a 3-KDS reductase to dihydrosphinganine (DHS). Serine palmitoyltransferase was recently shown to be essential for plant viability, but the 3...

  9. Enrichment of hydroxylated C24- and C26-acyl-chain sphingolipids mediates PIN2 apical sorting at trans-Golgi network subdomains

    PubMed Central

    Wattelet-Boyer, Valérie; Brocard, Lysiane; Jonsson, Kristoffer; Esnay, Nicolas; Joubès, Jérôme; Domergue, Frédéric; Mongrand, Sébastien; Raikhel, Natasha; Bhalerao, Rishikesh P.; Moreau, Patrick; Boutté, Yohann

    2016-01-01

    The post-Golgi compartment trans-Golgi Network (TGN) is a central hub divided into multiple subdomains hosting distinct trafficking pathways, including polar delivery to apical membrane. Lipids such as sphingolipids and sterols have been implicated in polar trafficking from the TGN but the underlying mechanisms linking lipid composition to functional polar sorting at TGN subdomains remain unknown. Here we demonstrate that sphingolipids with α-hydroxylated acyl-chains of at least 24 carbon atoms are enriched in secretory vesicle subdomains of the TGN and are critical for de novo polar secretory sorting of the auxin carrier PIN2 to apical membrane of Arabidopsis root epithelial cells. We show that sphingolipid acyl-chain length influences the morphology and interconnections of TGN-associated secretory vesicles. Our results uncover that the sphingolipids acyl-chain length links lipid composition of TGN subdomains with polar secretory trafficking of PIN2 to apical membrane of polarized epithelial cells. PMID:27681606

  10. Antisense inhibition of sorbitol synthesis leads to up-regulation of starch synthesis without altering CO2 assimilation in apple leaves.

    PubMed

    Cheng, Lailiang; Zhou, Rui; Reidel, Edwin J; Sharkey, Thomas D; Dandekar, Abhaya M

    2005-03-01

    Sorbitol is a primary end-product of photosynthesis in apple (Malus domestica Borkh.) and many other tree fruit species of the Rosaceae family. Sorbitol synthesis shares a common hexose phosphate pool with sucrose synthesis in the cytosol. In this study, 'Greensleeves' apple was transformed with a cDNA encoding aldose 6-phosphate reductase (A6PR, EC 1.1.1.200) in the antisense orientation. Antisense expression of A6PR decreased A6PR activity in mature leaves to approximately 15-30% of the untransformed control. The antisense plants had lower concentrations of sorbitol but higher concentrations of sucrose and starch in mature leaves at both dusk and predawn. (14)CO(2) pulse-chase labeling at ambient CO(2) demonstrated that partitioning of the newly fixed carbon to starch was significantly increased, whereas that to sucrose remained unchanged in the antisense lines with decreased sorbitol synthesis. Total activities of ribulose 1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39), sucrose-phosphate synthase (EC 2.4.1.14), and ADP-glucose pyrophosphorylase (EC 2.7.7.27) were not significantly altered in the antisense lines, whereas both stromal and cytosolic fructose-1,6-bisphosphatase (EC 3.1.3.11) activities were higher in the antisense lines with 15% of the control A6PR activity. Concentrations of glucose 6-phosphate and fructose 6-phosphate (F6P) were higher in the antisense plants than in the control, but the 3-phosphoglycerate concentration was lower in the antisense plants with 15% of the control A6PR activity. Fructose 2, 6-bisphosphate concentration increased in the antisense plants, but not to the extent expected from the increase in F6P, comparing sucrose-synthesizing species. There was no significant difference in CO(2) assimilation in response to photon flux density or intercellular CO(2) concentration. We concluded that cytosolic FBPase activity in vivo was down-regulated and starch synthesis was up-regulated in response to decreased sorbitol synthesis

  11. Effects of aging and life-prolonging diet on thyroid regulation of protein synthesis.

    PubMed

    Gromakova, I A; Konovalenko, O A

    2004-03-01

    The effect of thyroxin on the intensity of protein synthesis in rats of different age was studied during natural aging and in rats maintained on a low-caloric diet inhibiting aging. The intensity of protein synthesis decreased and the reaction to hormonal stimulus was absent in animals fed life-prolonging diet.

  12. PHOSPHATIDIC ACID PHOSPHOHYDROLASE1 and 2 Regulate Phospholipid Synthesis at the Endoplasmic Reticulum in Arabidopsis[W

    PubMed Central

    Eastmond, Peter J.; Quettier, Anne-Laure; Kroon, Johan T.M.; Craddock, Christian; Adams, Nicolette; Slabas, Antoni R.

    2010-01-01

    Phospholipid biosynthesis is essential for the construction of most eukaryotic cell membranes, but how this process is regulated in plants remains poorly understood. Here, we show that in Arabidopsis thaliana, two Mg2+-dependent phosphatidic acid phosphohydrolases called PAH1 and PAH2 act redundantly to repress phospholipid biosynthesis at the endoplasmic reticulum (ER). Leaves from pah1 pah2 double mutants contain ~1.8-fold more phospholipid than the wild type and exhibit gross changes in ER morphology, which are consistent with massive membrane overexpansion. The net rate of incorporation of [methyl-14C]choline into phosphatidylcholine (PC) is ~1.8-fold greater in the double mutant, and the transcript abundance of several key genes that encode enzymes involved in phospholipid synthesis is increased. In particular, we show that PHOSPHORYLETHANOLAMINE N-METHYLTRANSFERASE1 (PEAMT1) is upregulated at the level of transcription in pah1 pah2 leaves. PEAMT catalyzes the first committed step of choline synthesis in Arabidopsis and defines a variant pathway for PC synthesis not found in yeasts or mammals. Our data suggest that PAH1/2 play a regulatory role in phospholipid synthesis that is analogous to that described in Saccharomyces cerevisiae. However, the target enzymes differ, and key components of the signal transduction pathway do not appear to be conserved. PMID:20699392

  13. Involvement of the ribose operon repressor RbsR in regulation of purine nucleotide synthesis in Escherichia coli.

    PubMed

    Shimada, Tomohiro; Kori, Ayako; Ishihama, Akira

    2013-07-01

    Escherichia coli is able to utilize d-ribose as its sole carbon source. The genes for the transport and initial-step metabolism of d-ribose form a single rbsDACBK operon. RbsABC forms the ABC-type high-affinity d-ribose transporter, while RbsD and RbsK are involved in the conversion of d-ribose into d-ribose 5-phosphate. In the absence of inducer d-ribose, the ribose operon is repressed by a LacI-type transcription factor RbsR, which is encoded by a gene located downstream of this ribose operon. At present, the rbs operon is believed to be the only target of regulation by RbsR. After Genomic SELEX screening, however, we have identified that RbsR binds not only to the rbs promoter but also to the promoters of a set of genes involved in purine nucleotide metabolism. Northern blotting analysis indicated that RbsR represses the purHD operon for de novo synthesis of purine nucleotide but activates the add and udk genes involved in the salvage pathway of purine nucleotide synthesis. Taken together, we propose that RbsR is a global regulator for switch control between the de novo synthesis of purine nucleotides and its salvage pathway.

  14. Extensive sphingolipid depletion does not affect lipid raft integrity or lipid raft localization and efflux function of the ABC transporter MRP1

    PubMed Central

    Klappe, Karin; Dijkhuis, Anne-Jan; Hummel, Ina; vanDam, Annie; Ivanova, Pavlina T.; Milne, Stephen B.; Myers, David S.; Brown, H. Alex; Permentier, Hjalmar; Kok, Jan W.

    2013-01-01

    We show that highly efficient depletion of sphingolipids in two different cell lines does not abrogate the ability to isolate Lubrol-based DRMs (detergent-resistant membranes) or detergent-free lipid rafts from these cells. Compared with control, DRM/detergent-free lipid raft fractions contain equal amounts of protein, cholesterol and phospholipid, whereas the classical DRM/lipid raft markers Src, caveolin-1 and flotillin display the same gradient distribution. DRMs/detergent-free lipid rafts themselves are severely depleted of sphingolipids. The fatty acid profile of the remaining sphingolipids as well as that of the glycerophospholipids shows several differences compared with control, most prominently an increase in highly saturated C16 species. The glycerophospholipid headgroup composition is unchanged in sphingolipid-depleted cells and cell-derived detergent-free lipid rafts. Sphingolipid depletion does not alter the localization of MRP1 (multidrug-resistance-related protein 1) in DRMs/detergent-free lipid rafts or MRP1-mediated efflux of carboxyfluorescein. We conclude that extensive sphingolipid depletion does not affect lipid raft integrity in two cell lines and does not affect the function of the lipid-raft-associated protein MRP1. PMID:20604746

  15. Down-regulation of sorbitol dehydrogenase and up-regulation of sucrose synthase in shoot tips of the transgenic apple trees with decreased sorbitol synthesis.

    PubMed

    Zhou, Rui; Cheng, Lailiang; Dandekar, Abhaya M

    2006-01-01

    Both sorbitol and sucrose are translocated to, and utilized in, sink tissues of apple (Malus domestica). Considering that antisense suppression of aldose 6-phosphate reductase resulted in lower concentrations of sorbitol and higher concentrations of sucrose in source leaves without altering the vegetative growth of apple trees, it was hypothesized that sorbitol metabolism is down-regulated and sucrose metabolism is up-regulated in shoot tips of the transgenic plants. Carbohydrate measurements indicated that sorbitol concentration was lower whereas sucrose concentration was higher in the shoot tips of transgenic apple plants with decreased sorbitol synthesis compared with the untransformed control. However, the shoot relative growth rate was not altered in the transgenic plants. Sorbitol dehydrogenase (SDH) activity was decreased; acid invertase activity and neutral invertase activity remained the same, whereas sucrose synthase (SUSY) activity was increased in shoot tips of the transgenic plants. The SDH transcript level was lower whereas the SUSY transcript level was higher in shoot tips of the transgenic plants. SDH activity and SDH transcript level were specifically stimulated by exogenous sorbitol fed to the shoot tips via the transpiration stream but were specifically inhibited by sucrose. SUSY activity and SUSY transcript level were dramatically enhanced by sucrose, but decreased by glucose and fructose. Neither acid invertase nor neutral invertase activity responded to sucrose, glucose, fructose, or any other sugars tested. It is concluded that sorbitol dehydrogenase is down-regulated, whereas sucrose synthase is up-regulated in shoot tips of the transgenic apple trees with decreased sorbitol synthesis, leading to homeostasis of vegetative growth. Sorbitol and sucrose act as signal molecules to modulate the expression and activities of sorbitol dehydrogenase and sucrose synthase, both of which play an important role in determining the sink strength of apple

  16. Feeding a Modified Fish Diet to Bottlenose Dolphins Leads to an Increase in Serum Adiponectin and Sphingolipids

    PubMed Central

    Sobolesky, Philip M.; Harrell, Tyler S.; Parry, Celeste; Venn-Watson, Stephanie; Janech, Michael G.

    2016-01-01

    Feeding a modified fish diet has been suggested to improve insulin sensitivity in bottlenose dolphins; however, insulin sensitivity was not directly measured. Since demonstrating an improvement in insulin sensitivity is technically difficult in dolphins, we postulated that directional changes in the hormone axis: fibroblast growth factor 21 (FGF21)/Adiponectin/Ceramide (Cer), could provide further support to this hypothesis. We measured 2-h post-prandial serum FGF21, total adiponectin, percent unmodified adiponectin, ceramide, and sphingosine levels from dolphins fed a diet rich in heptadecanoic acid (C17:0) over 24 weeks. Serum FGF21 was quantified by ELISA with an observed range of 129–1599 pg/ml, but did not significantly change over the 24-week study period. Total adiponectin levels (mean ± SD) significantly increased from 776 ± 400 pmol/ml at week 0 to 1196 ± 467 pmol/ml at week 24. The percent unmodified adiponectin levels (mean ± SD) decreased from 23.8 ± 6.0% at week 0 to 15.2 ± 5.2% at week 24. Interestingly, although FGF21 levels did not change, there was a good correlation between FGF21 and total adiponectin (ρ = 0.788, P < 0.001). We quantified the abundances of serum ceramides and sphingosines (SPH) because adiponectin has a defined role in sphingolipid metabolism through adiponectin receptor-mediated activation of ceramidases. The most abundant ceramide in dolphin sera was Cer 24:1 comprising 49% of the ceramides measured. Significant reductions were observed in the unsaturated Cer 18:1, Cer 20:1, and Cer 24:1, whereas significant increases were observed in saturated Cer 22:0, Cer 24:0, and Cer 26:0. However, total serum ceramides did not change. Significant elevations were detected for total sphingosine, dihydrosphingosine, sphingosine-1-phosphate, and dihydrosphingosine-1-phosphate. Proteomic analysis of the serum proteins revealed few changes in serum proteins over the study period. In conclusion

  17. Altering sphingolipid composition with aging induces contractile dysfunction of gastric smooth muscle via K(Ca) 1.1 upregulation.

    PubMed

    Choi, Shinkyu; Kim, Ji Aee; Kim, Tae Hun; Li, Hai-Yan; Shin, Kyong-Oh; Lee, Yong-Moon; Oh, Seikwan; Pewzner-Jung, Yael; Futerman, Anthony H; Suh, Suk Hyo

    2015-12-01

    K(Ca) 1.1 regulates smooth muscle contractility by modulating membrane potential, and age-associated changes in K(Ca) 1.1 expression may contribute to the development of motility disorders of the gastrointestinal tract. Sphingolipids (SLs) are important structural components of cellular membranes whose altered composition may affect K(Ca) 1.1 expression. Thus, in this study, we examined whether altered SL composition due to aging may affect the contractility of gastric smooth muscle (GSM). We studied changes in ceramide synthases (CerS) and SL levels in the GSM of mice of varying ages and compared them with those in young CerS2-null mice. The levels of C16- and C18-ceramides, sphinganine, sphingosine, and sphingosine 1-phosphate were increased, and levels of C22, C24:1 and C24 ceramides were decreased in the GSM of both aged wild-type and young CerS2-null mice. The altered SL composition upregulated K(Ca) 1.1 and increased K(Ca) 1.1 currents, while no change was observed in K(Ca) 1.1 channel activity. The upregulation of KC a 1.1 impaired intracellular Ca²⁺mobilization and decreased phosphorylated myosin light chain levels, causing GSM contractile dysfunction. Additionally, phosphoinositide 3-kinase, protein kinase Cζ , c-Jun N-terminal kinases, and nuclear factor kappa-B were found to be involved in K(Ca) 1.1 upregulation. Our findings suggest that age-associated changes in SL composition or CerS2 ablation upregulate K(Ca) 1.1 via the phosphoinositide 3-kinase/protein kinase Cζ /c-Jun N-terminal kinases/nuclear factor kappa-B-mediated pathway and impair Ca²⁺ mobilization, which thereby induces the contractile dysfunction of GSM. CerS2-null mice exhibited similar effects to aged wild-type mice; therefore, CerS2-null mouse models may be utilized for investigating the pathogenesis of aging-associated motility disorders.

  18. Could thiamine pyrophosphate be a regulator of the nitric oxide synthesis in the endothelial cell of diabetic patients?

    PubMed

    Alcázar-Leyva, Susana; Alvarado-Vásquez, Noé

    2011-05-01

    Thiamine (Vitamin B1) is considered an essential micronutrient for humans; its deficient intake brings about the Wernicke-Korsakoff syndrome (encephalopathy and psychosis) or beriberi (a neurological and cardiovascular disease). Once thiamine enters the cells it is phosphorylated by thiamine pyrophosphokinase (TPPK), and converted into the coenzyme thiamine pyrophosphate (TPP), the active form of thiamine. TPP is a relevant cofactor for transketolase (TK), α-ketoglutarate dehydrogenase (αKDH), and pyruvate dehydrogenase (PDH), all these enzymes are fundamental for glucose metabolism. Diabetes mellitus (DM), however, is considered both a deficient thiamine and deficient energy state, as a consequence of the limited TPP synthesis. Recent evidences have shown that the administration of thiamine or lipid-soluble derivatives, such as benfotiamine (developed to improve the bioavailability of thiamine), has positive effects in the diabetic patient (after thiamine is transformed into TPP). For this reason, administration of supplements with TPP in the diabetic patients is recommended to avoid complications, like neuropathy and nephropathy. It has been suggested that these beneficial effects are a consequence of the activation of TK (pentose pathway) or the PDH complex in mitochondria. Nitric oxide (NO) is synthesized by the endothelial cell and is also an important element for the viability and functionality of this cell type. However, in the DM patient, a deficient synthesis of NO has been reported. It is relevant to mention that recent evidences have led to propose mitochondrial activity as an important regulator of nitric oxide synthesis (ON). We consider that the exogenous administration of TPP facilitates the utilization of this molecule, regulating some metabolic processes such as phosphorylation of thiamine by TPPK, energy consumption (ATP), as well as mitochondrial activity, inducing eventually NO synthesis. If this is confirmed, the administration of TPP to the

  19. Six1 induces protein synthesis signaling expression in duck myoblasts mainly via up-regulation of mTOR

    PubMed Central

    Wang, Haohan; Li, Xinxin; Liu, Hehe; Sun, Lingli; Zhang, Rongping; Li, Liang; Wangding, Mincheng; Wang, Jiwen

    2016-01-01

    Abstract As a critical transcription factor, Six1 plays an important role in the regulation of myogenesis and muscle development. However, little is known about its regulatory mechanism associated with muscular protein synthesis. The objective of this study was to investigate the effects of overexpression ofSix1 on the expression of key protein metabolism-related genes in duck myoblasts. Through an experimental model where duck myoblasts were transfected with a pEGFP-duSix1 construct, we found that overexpression of duckSix1 could enhance cell proliferation activity and increase mRNA expression levels of key genes involved in the PI3K/Akt/mTOR signaling pathway, while the expression of FOXO1, MuRF1and MAFbx was not significantly altered, indicating thatSix1 could promote protein synthesis in myoblasts through up-regulating the expression of several related genes. Additionally, in duck myoblasts treated with LY294002 and rapamycin, the specific inhibitors ofPI3K and mTOR, respectively, the overexpression of Six1 could significantly ameliorate inhibitive effects of these inhibitors on protein synthesis. Especially, the mRNA expression levels of mTOR and S6K1 were observed to undergo a visible change, and a significant increase in protein expression of S6K1 was seen. These data suggested that Six1plays an important role in protein synthesis, which may be mainly due to activation of the mTOR signaling pathway. PMID:27007909

  20. Expression of the Saccharomyces cerevisiae inositol-1-phosphate synthase (INO1) gene is regulated by factors that affect phospholipid synthesis.

    PubMed Central

    Hirsch, J P; Henry, S A

    1986-01-01

    The INO1 gene of Saccharomyces cerevisiae encodes the regulated enzyme inositol-1-phosphate synthase, which catalyzes the first committed step in the synthesis of inositol-containing phospholipids. The expression of this gene was analyzed under conditions known to regulate phospholipid synthesis. RNA blot hybridization with a genomic clone for INO1 detected two RNA species of 1.8 and 0.6 kb. The abundance of the 1.8-kb RNA was greatly decreased when the cells were grown in the presence of the phospholipid precursor inositol, as was the enzyme activity of the synthase. Complementation analysis showed that this transcript encoded the INO1 gene product. The level of INO1 RNA was repressed 12-fold when the cells were grown in medium containing inositol, and it was repressed 33-fold when the cells were grown in the presence of inositol and choline together. The INO1 transcript was present at a very low level in cells containing mutations (ino2 and ino4) in regulatory genes unlinked to INO1 that result in inositol auxotrophy. The transcript was constitutively overproduced in cells containing a mutation (opi1) that causes constitutive expression of inositol-1-phosphate synthase and results in excretion of inositol. The expression of INO1 RNA was also examined in cells containing a mutation (cho2) affecting the synthesis of phosphatidylcholine. In contrast to what was observed in wild-type cells, growth of cho2 cells in medium containing inositol did not result in a significant decrease in INO1 RNA abundance. Inositol and choline together were required for repression of the INO1 transcript in these cells, providing evidence for a regulatory link between the synthesis of inositol- and choline-containing lipids. The level of the 0.6-kb RNA was affected, although to a lesser degree, by many of the same factors that influence INO1 expression. Images PMID:3025587

  1. Alkaline Ceramidase 3 Deficiency Results in Purkinje Cell Degeneration and Cerebellar Ataxia Due to Dyshomeostasis of Sphingolipids in the Brain

    PubMed Central

    Preston, Chet; Wang, Louis; Yi, Jae Kyo; Lin, Chih-Li; Sun, Wei; Spyropoulos, Demetri D.; Rhee, Soyoung; Li, Mingsong; Zhou, Jie; Ge, Shaoyu; Zhang, Guofeng; Snider, Ashley J.; Hannun, Yusuf A.; Obeid, Lina M.; Mao, Cungui

    2015-01-01

    Dyshomeostasis of both ceramides and sphingosine-1-phosphate (S1P) in the brain has been implicated in aging-associated neurodegenerative disorders in humans. However, mechanisms that maintain the homeostasis of these bioactive sphingolipids in the brain remain unclear. Mouse alkaline ceramidase 3 (Acer3), which preferentially catalyzes the hydrolysis of C18:1-ceramide, a major unsaturated long-chain ceramide species in the brain, is upregulated with age in the mouse brain. Acer3 knockout causes an age-dependent accumulation of various ceramides and C18:1-monohexosylceramide and abolishes the age-related increase in the levels of sphingosine and S1P in the brain; thereby resulting in Purkinje cell degeneration in the cerebellum and deficits in motor coordination and balance. Our results indicate that Acer3 plays critically protective roles in controlling the homeostasis of various sphingolipids, including ceramides, sphingosine, S1P, and certain complex sphingolipids in the brain and protects Purkinje cells from premature degeneration. PMID:26474409

  2. Nutritional and hormonal regulation of malic enzyme synthesis in rat mammary gland.

    PubMed Central

    Lobato, M F; Ros, M; Moreno, F J; García-Ruíz, J P

    1986-01-01

    Cytosolic malic enzyme was purified from rat mammary gland by L-malate affinity chromatography. The pure enzyme obtained was used to produce a specific antiserum in a rabbit. Relative synthesis of malic enzyme in the mammary gland of mid-lactating rats was 0.097%, measured by labelling the enzyme in isolated acini. When food was removed, malic enzyme synthesis decreased to 35% and 20% of the control value at 4 and 6 h respectively. Incorporation of [3H]leucine into soluble proteins was constant during the first 6 h of starvation. When lactating rats (maintained with their pups) were starved for 24 h and then re-fed, the relative rate of enzyme synthesis increased 2.5-, 4-, and 4.5-fold at 3 h, 6 h and 18 h respectively after initiation of re-feeding. The relative rate of malic enzyme synthesis was about 50% of normal at 15 h after weaning, whereas the rate of synthesis of soluble proteins did not change. Administration of bromocriptine or adrenalectomy of lactating rats decreased the relative rate of synthesis of malic enzyme by 40% or 30% respectively; these effects were counteracted by hormone supplementation. Hormone therapy also caused an increase in the rate of incorporation of [3H]leucine into soluble proteins and in malic enzyme activity. Images Fig. 1. PMID:3753458

  3. Global negative regulation of Streptomyces coelicolor antibiotic synthesis mediated by an absA-encoded putative signal transduction system.

    PubMed Central

    Brian, P; Riggle, P J; Santos, R A; Champness, W C

    1996-01-01

    Streptomycete antibiotic synthesis is coupled to morphological differentiation such that antibiotics are produced as a colony sporulates. Streptomyces coelicolor produces several structurally and genetically distinct antibiotics. The S. coelicolor absA locus was defined by four UV-induced mutations that globally blocked antibiotic biosynthesis without blocking morphological differentiation. We show that the absA locus encodes a putative eubacterial two-component sensor kinase-response regulator system. All four mutations lie within a single open reading frame, designated absA1, which is predicted to encode a sensor histidine kinase. A second gene downstream of absA1, absA2, is predicted to encode the cognate response regulator. In marked contrast to the antibiotic-deficient phenotype of the previously described absA mutants, the phenotype caused by disruption mutations in the absA locus is precocious hyperproduction of the antibiotics actinorhodin and undecylprodigiosin. Precocious hyperproduction of these antibiotics is correlated with premature expression of XylE activity in a transcriptional fusion to an actinorhodin biosynthetic gene. We propose that the absA locus encodes a signal transduction mechanism that negatively regulates synthesis of the multiple antibiotics produced by S. coelicolor. PMID:8655502

  4. Root jasmonic acid synthesis and perception regulate folivore-induced shoot metabolites and increase Nicotiana attenuata resistance.

    PubMed

    Fragoso, Variluska; Rothe, Eva; Baldwin, Ian T; Kim, Sang-Gyu

    2014-06-01

    While jasmonic acid (JA) signaling is widely accepted as mediating plant resistance to herbivores, and the importance of the roots in plant defenses is recently being recognized, the role of root JA in the defense of above-ground parts remains unstudied. To restrict JA impairment to the roots, we micrografted wildtype Nicotiana attenuata shoots to the roots of transgenic plants impaired in JA signaling and evaluated ecologically relevant traits in the glasshouse and in nature. Root JA synthesis and perception are involved in regulating nicotine production in roots. Strikingly, systemic root JA regulated local leaf JA and abscisic acid (ABA) concentrations, which were associated with differences in nicotine transport from roots to leaves via the transpiration stream. Root JA signaling also regulated the accumulation of other shoot metabolites; together these account for differences in resistance against a generalist, Spodoptera littoralis, and a specialist herbivore, Manduca sexta. In N. attenuata's native habitat, silencing root JA synthesis increased the shoot damage inflicted by Empoasca leafhoppers, which are able to select natural jasmonate mutants. Silencing JA perception in roots also increased damage by Tupiocoris notatus. We conclude that attack from above-ground herbivores recruits root JA signaling to launch the full complement of plant defense responses.

  5. FLOURY ENDOSPERM7 encodes a regulator of starch synthesis and amyloplast development essential for peripheral endosperm development in rice

    PubMed Central

    Zhang, Long; Ren, Yulong; Lu, Bingyue; Yang, Chunyan; Feng, Zhiming; Liu, Zhou; Chen, Jun; Ma, Weiwei; Wang, Ying; Yu, Xiaowen; Wang, Yunlong; Zhang, Wenwei; Wang, Yihua; Liu, Shijia; Wu, Fuqing; Zhang, Xin; Guo, Xiuping; Bao, Yiqun; Jiang, Ling; Wan, Jianmin

    2016-01-01

    In cereal crops, starch synthesis and storage depend mainly on a specialized class of plastids, termed amyloplasts. Despite the importance of starch, the molecular machinery regulating starch synthesis and amyloplast development remains largely unknown. Here, we report the characterization of the rice (Oryza sativa) floury endosperm7 (flo7) mutant, which develops a floury-white endosperm only in the periphery and not in the inner portion. Consistent with the phenotypic alternation in flo7 endosperm, the flo7 mutant had reduced amylose content and seriously disrupted amylopectin structure only in the peripheral endosperm. Notably, flo7 peripheral endosperm cells showed obvious defects in compound starch grain development. Map-based cloning of FLO7 revealed that it encodes a protein of unknown function. FLO7 harbors an N-terminal transit peptide capable of targeting functional FLO7 fused to green fluorescent protein to amyloplast stroma in developing endosperm cells, and a domain of unknown function 1338 (DUF1338) that is highly conserved in green plants. Furthermore, our combined β-glucuronidase activity and RNA in situ hybridization assays showed that the FLO7 gene was expressed ubiquitously but exhibited a specific expression in the endosperm periphery. Moreover, a set of in vivo experiments demonstrated that the missing 32 aa in the flo7 mutant protein are essential for the stable accumulation of FLO7 in the endosperm. Together, our findings identify FLO7 as a unique plant regulator required for starch synthesis and amyloplast development within the peripheral endosperm and provide new insights into the spatial regulation of endosperm development in rice. PMID:26608643

  6. FLOURY ENDOSPERM7 encodes a regulator of starch synthesis and amyloplast development essential for peripheral endosperm development in rice.

    PubMed

    Zhang, Long; Ren, Yulong; Lu, Bingyue; Yang, Chunyan; Feng, Zhiming; Liu, Zhou; Chen, Jun; Ma, Weiwei; Wang, Ying; Yu, Xiaowen; Wang, Yunlong; Zhang, Wenwei; Wang, Yihua; Liu, Shijia; Wu, Fuqing; Zhang, Xin; Guo, Xiuping; Bao, Yiqun; Jiang, Ling; Wan, Jianmin

    2016-02-01

    In cereal crops, starch synthesis and storage depend mainly on a specialized class of plastids, termed amyloplasts. Despite the importance of starch, the molecular machinery regulating starch synthesis and amyloplast development remains largely unknown. Here, we report the characterization of the rice (Oryza sativa) floury endosperm7 (flo7) mutant, which develops a floury-white endosperm only in the periphery and not in the inner portion. Consistent with the phenotypic alternation in flo7 endosperm, the flo7 mutant had reduced amylose content and seriously disrupted amylopectin structure only in the peripheral endosperm. Notably, flo7 peripheral endosperm cells showed obvious defects in compound starch grain development. Map-based cloning of FLO7 revealed that it encodes a protein of unknown function. FLO7 harbors an N-terminal transit peptide capable of targeting functional FLO7 fused to green fluorescent protein to amyloplast stroma in developing endosperm cells, and a domain of unknown function 1338 (DUF1338) that is highly conserved in green plants. Furthermore, our combined β-glucuronidase activity and RNA in situ hybridization assays showed that the FLO7 gene was expressed ubiquitously but exhibited a specific expression in the endosperm periphery. Moreover, a set of in vivo experiments demonstrated that the missing 32 aa in the flo7 mutant protein are essential for the stable accumulation of FLO7 in the endosperm. Together, our findings identify FLO7 as a unique plant regulator required for starch synthesis and amyloplast development within the peripheral endosperm and provide new insights into the spatial regulation of endosperm development in rice.

  7. DHA-mediated enhancement of TRAIL-induced apoptosis in colon cancer cells is associated with engagement of mitochondria and specific alterations in sphingolipid metabolism.

    PubMed

    Skender, Belma; Hofmanová, Jiřina; Slavík, Josef; Jelínková, Iva; Machala, Miroslav; Moyer, Mary Pat; Kozubík, Alois; Hyršlová Vaculová, Alena

    2014-09-01

    Docosahexaenoic acid (DHA), an n-3 polyunsaturated fatty acid present in fish oil, may exert cytotoxic and/or cytostatic effects on colon cancer cells when applied individually or in combination with some anticancer drugs. Here we demonstrate a selective ability of subtoxic doses of DHA to enhance antiproliferative and apoptotic effects of clinically useful cytokine TRAIL (tumor necrosis factor-related apoptosis inducing ligand) in cancer but not normal human colon cells. DHA-mediated stimulation of TRAIL-induced apoptosis was associated with extensive engagement of mitochondrial pathway (Bax/Bak activation, drop of mitochondrial membrane potential, cytochrome c release), activation of endoplasmic reticulum stress response (CHOP upregulation, changes in PERK level), decrease of cellular inhibitor of apoptosis protein (XIAP, cIAP1) levels and significant changes in sphingolipid metabolism (intracellular levels of ceramides, hexosyl ceramides, sphingomyelines, sphingosines; HPLC/MS/MS). Interestingly, we found significant differences in representation of various classes of ceramides (especially C16:0, C24:1) between the cancer and normal colon cells treated with DHA and TRAIL, and suggested their potential role in the regulation of the cell response to the drug combination. These study outcomes highlight the potential of DHA for a new combination therapy with TRAIL for selective elimination of colon cancer cells via simultaneous targeting of multiple steps in apoptotic pathways.

  8. Revisiting Plant Plasma Membrane Lipids in Tobacco: A Focus on Sphingolipids1

    PubMed Central

    Cacas, Jean-Luc; Grosjean, Kevin; Gerbeau-Pissot, Patricia; Lherminier, Jeannine; Rombouts, Yoann; Maes, Emmanuel; Gronnier, Julien; Furt, Fabienne; Fouillen, Laetitia; Bayer, Emmanuelle; Cluzet, Stéphanie; Schmitter, Jean-Marie; Deleu, Magali; Lins, Laurence; Simon-Plas, Françoise; Mongrand, Sébastien

    2016-01-01

    The lipid composition of plasma membrane (PM) and the corresponding detergent-insoluble membrane (DIM) fraction were analyzed with a specific focus on highly polar sphingolipids, so-called glycosyl inositol phosphorylceramides (GIPCs). Using tobacco (Nicotiana tabacum) ‘Bright Yellow 2’ cell suspension and leaves, evidence is provided that GIPCs represent up to 40 mol % of the PM lipids. Comparative analysis of DIMs with the PM showed an enrichment of 2-hydroxylated very-long-chain fatty acid-containing GIPCs and polyglycosylated GIPCs in the DIMs. Purified antibodies raised against these GIPCs were further used for immunogold-electron microscopy strategy, revealing the distribution of polyglycosylated GIPCs in domains of 35 ± 7 nm in the plane of the PM. Biophysical studies also showed strong interactions between GIPCs and sterols and suggested a role for very-long-chain fatty acids in the interdigitation between the two PM-composing monolayers. The ins and outs of lipid asymmetry, raft formation, and interdigitation in plant membrane biology are finally discussed. PMID:26518342

  9. Inactivation of Ceramide Synthase 6 in Mice Results in an Altered Sphingolipid Metabolism and Behavioral Abnormalities*

    PubMed Central

    Ebel, Philipp; vom Dorp, Katharina; Petrasch-Parwez, Elisabeth; Zlomuzica, Armin; Kinugawa, Kiyoka; Mariani, Jean; Minich, David; Ginkel, Christina; Welcker, Jochen; Degen, Joachim; Eckhardt, Matthias; Dere, Ekrem; Dörmann, Peter; Willecke, Klaus

    2013-01-01

    The N-acyl chain length of ceramides is determined by the specificity of different ceramide synthases (CerS). The CerS family in mammals consists of six members with different substrate specificities and expression patterns. We have generated and characterized a mouse line harboring an enzymatically inactive ceramide synthase 6 (CerS6KO) gene and lacz reporter cDNA coding for β-galactosidase directed by the CerS6 promoter. These mice display a decrease in C16:0 containing sphingolipids. Relative to wild type tissues the amount of C16:0 containing sphingomyelin in kidney is ∼35%, whereas we find a reduction of C16:0 ceramide content in the small intestine to about 25%. The CerS6KO mice show behavioral abnormalities including a clasping abnormality of their hind limbs and a habituation deficit. LacZ reporter expression in the brain reveals CerS6 expression in hippocampus, cortex, and the Purkinje cell layer of the cerebellum. Using newly developed antibodies that specifically recognize the CerS6 protein we show that the endogenous CerS6 protein is N-glycosylated and expressed in several tissues of mice, mainly kidney, small and large intestine, and brain. PMID:23760501

  10. Inactivation of ceramide synthase 6 in mice results in an altered sphingolipid metabolism and behavioral abnormalities.

    PubMed

    Ebel, Philipp; Vom Dorp, Katharina; Petrasch-Parwez, Elisabeth; Zlomuzica, Armin; Kinugawa, Kiyoka; Mariani, Jean; Minich, David; Ginkel, Christina; Welcker, Jochen; Degen, Joachim; Eckhardt, Matthias; Dere, Ekrem; Dörmann, Peter; Willecke, Klaus

    2013-07-19

    The N-acyl chain length of ceramides is determined by the specificity of different ceramide synthases (CerS). The CerS family in mammals consists of six members with different substrate specificities and expression patterns. We have generated and characterized a mouse line harboring an enzymatically inactive ceramide synthase 6 (CerS6KO) gene and lacz reporter cDNA coding for β-galactosidase directed by the CerS6 promoter. These mice display a decrease in C16:0 containing sphingolipids. Relative to wild type tissues the amount of C16:0 containing sphingomyelin in kidney is ∼35%, whereas we find a reduction of C16:0 ceramide content in the small intestine to about 25%. The CerS6KO mice show behavioral abnormalities including a clasping abnormality of their hind limbs and a habituation deficit. LacZ reporter expression in the brain reveals CerS6 expression in hippocampus, cortex, and the Purkinje cell layer of the cerebellum. Using newly developed antibodies that specifically recognize the CerS6 protein we show that the endogenous CerS6 protein is N-glycosylated and expressed in several tissues of mice, mainly kidney, small and large intestine, and brain.

  11. Digestion of Ceramide 2-Aminoethylphosphonate, a Sphingolipid from the Jumbo Flying Squid Dosidicus gigas, in Mice.

    PubMed

    Tomonaga, Nami; Manabe, Yuki; Sugawara, Tatsuya

    2017-04-01

    Ceramide 2-aminoethylphosphonate (CAEP), a sphingophosphonolipid containing a carbon-phosphorus bond, is frequently found in marine organisms and has a unique triene type of sphingoid base in its structure. CAEP has not been evaluated as a food ingredient, although it is generally contained in Mollusca organisms such as squids and shellfish, which are consumed worldwide. In this study, we aimed to elucidate the effects of CAEP as a food component by evaluating the digestion of CAEP extracted from the skin of the jumbo flying squid Dosidicus gigas. Our results revealed that dietary CAEP was digested to free sphingoid bases via ceramides by the mouse small intestinal mucosa. At pH 7.2, CAEP was hydrolyzed more rapidly than the major mammalian sphingolipid sphingomyelin; however, the hydrolysis of CAEP was similar to that of sphingomyelin at pH 9.0. Thus, the digestion of CAEP may be catalyzed by alkaline spingomyelinase and other enzymes. Our findings provide important insights into the digestion of the dietary sphingophosphonolipid CAEP in marine foods.

  12. A minimalist approach to MALDI imaging of glycerophospholipids and sphingolipids in rat brain sections

    NASA Astrophysics Data System (ADS)

    Wang, Hay-Yan J.; Post, Shelley N. Jackson Jeremy; Woods, Amina S.

    2008-12-01

    Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a powerful tool that has allowed researchers to directly probe tissue molecular structure and drug content with minimal manipulations, while maintaining anatomical integrity. In the present work glycerophospholipids and sphingolipids images were acquired from 16-[mu]m thick coronal rat brain sections using MALDI-MS. Images of phosphatidylinositol 38:4 (PI 38:4), sulfatide 24:1 (ST 24:1), and hydroxyl sulfatide 24:1 (ST 24:1 (OH)) were acquired in negative ion mode, while the images of phosphatidylcholine 34:1 (PC 34:1), potassiated phosphatidylcholines 32:0 (PC 32:0 + K+) and 36:1 (PC 36:1 + K+) were acquired in positive ion mode. The images of PI 38:4 and PC 36:1 + K+ show the preferential distribution of these two lipids in gray matter; and the images of two sulfatides and PC 32:0 + K+ show their preferential distribution in white matter. In addition, the gray cortical band and its adjacent anatomical structures were also identified by contrasting their lipid makeup. The resulting images were compared to lipid images acquired by secondary ion mass spectrometry (SIMS). The suitability of TLC sprayers, Collison Nebulizer, and artistic airbrush were also evaluated as means for matrix deposition.

  13. From Molecules to the Clinic: Linking Schizophrenia and Metabolic Syndrome through Sphingolipids Metabolism.

    PubMed

    Castillo, Rolando I; Rojo, Leonel E; Henriquez-Henriquez, Marcela; Silva, Hernán; Maturana, Alejandro; Villar, María J; Fuentes, Manuel; Gaspar, Pablo A

    2016-01-01

    Metabolic syndrome (MS) is a prevalent and severe comorbidity observed in schizophrenia (SZ). The exact nature of this association is controversial and very often accredited to the effects of psychotropic medications and disease-induced life-style modifications, such as inactive lifestyle, poor dietary choices, and smoking. However, drug therapy and disease-induced lifestyle factors are likely not the only factors contributing to the observed converging nature of these conditions, since an increased prevalence of MS is also observed in first episode and drug-naïve psychosis populations. MS and SZ share common intrinsic susceptibility factors and etiopathogenic mechanisms, which may change the way we approach clinical management of SZ patients. Among the most relevant common pathogenic pathways of SZ and MS are alterations in the sphingolipids (SLs) metabolism and SLs homeostasis. SLs have important structural functions as they participate in the formation of membrane "lipid rafts." SLs also play physiological roles in cell differentiation, proliferation, and inflammatory processes, which might be part of MS/SZ common pathophysiological processes. In this article we review a plausible mechanism to explain the link between MS and SZ through a disruption in SL homeostasis. Additionally, we provide insights on how this hypothesis can lead to the developing of new diagnostic/therapeutic technologies for SZ patients.

  14. Bladder cancer cell growth and motility implicate cannabinoid 2 receptor-mediated modifications of sphingolipids metabolism

    PubMed Central

    Bettiga, Arianna; Aureli, Massimo; Colciago, Giorgia; Murdica, Valentina; Moschini, Marco; Lucianò, Roberta; Canals, Daniel; Hannun, Yusuf; Hedlund, Petter; Lavorgna, Giovanni; Colombo, Renzo; Bassi, Rosaria; Samarani, Maura; Montorsi, Francesco; Salonia, Andrea; Benigni, Fabio

    2017-01-01

    The inhibitory effects demonstrated by activation of cannabinoid receptors (CB) on cancer proliferation and migration may also play critical roles in controlling bladder cancer (BC). CB expression on human normal and BC specimens was tested by immunohistochemistry. Human BC cells RT4 and RT112 were challenged with CB agonists and assessed for proliferation, apoptosis, and motility. Cellular sphingolipids (SL) constitution and metabolism were evaluated after metabolic labelling. CB1-2 were detected in BC specimens, but only CB2 was more expressed in the tumour. Both cell lines expressed similar CB2. Exposure to CB2 agonists inhibited BC growth, down-modulated Akt, induced caspase 3-activation and modified SL metabolism. Baseline SL analysis in cell lines showed differences linked to unique migratory behaviours and cytoskeletal re-arrangements. CB2 activation changed the SL composition of more aggressive RT112 cells by reducing (p < 0.01) Gb3 ganglioside (−50 ± 3%) and sphingosine 1-phosphate (S1P, −40 ± 4%), which ended up to reduction in cell motility (−46 ± 5%) with inhibition of p-SRC. CB2-selective antagonists, gene silencing and an inhibitor of SL biosynthesis partially prevented CB2 agonist-induced effects on cell viability and motility. CB2 activation led to ceramide-mediated BC cell apoptosis independently of SL constitutive composition, which instead was modulated by CB2 agonists to reduce cell motility. PMID:28191815

  15. From Molecules to the Clinic: Linking Schizophrenia and Metabolic Syndrome through Sphingolipids Metabolism

    PubMed Central

    Castillo, Rolando I.; Rojo, Leonel E.; Henriquez-Henriquez, Marcela; Silva, Hernán; Maturana, Alejandro; Villar, María J.; Fuentes, Manuel; Gaspar, Pablo A.

    2016-01-01

    Metabolic syndrome (MS) is a prevalent and severe comorbidity observed in schizophrenia (SZ). The exact nature of this association is controversial and very often accredited to the effects of psychotropic medications and disease-induced life-style modifications, such as inactive lifestyle, poor dietary choices, and smoking. However, drug therapy and disease-induced lifestyle factors are likely not the only factors contributing to the observed converging nature of these conditions, since an increased prevalence of MS is also observed in first episode and drug-naïve psychosis populations. MS and SZ share common intrinsic susceptibility factors and etiopathogenic mechanisms, which may change the way we approach clinical management of SZ patients. Among the most relevant common pathogenic pathways of SZ and MS are alterations in the sphingolipids (SLs) metabolism and SLs homeostasis. SLs have important structural functions as they participate in the formation of membrane “lipid rafts.” SLs also play physiological roles in cell differentiation, proliferation, and inflammatory processes, which might be part of MS/SZ common pathophysiological processes. In this article we review a plausible mechanism to explain the link between MS and SZ through a disruption in SL homeostasis. Additionally, we provide insights on how this hypothesis can lead to the developing of new diagnostic/therapeutic technologies for SZ patients. PMID:27877101

  16. Fast screening of highly glycosylated plant sphingolipids by tandem mass spectrometry.

    PubMed

    Buré, Corinne; Cacas, Jean-Luc; Wang, Fen; Gaudin, Karen; Domergue, Frédéric; Mongrand, Sébastien; Schmitter, Jean-Marie

    2011-10-30

    The structural characterization of Glycosyl-Inositol-Phospho-Ceramides (GIPCs), which are the main sphingolipids of plant tissues, is a critical step towards the understanding of their physiological function. After optimization of their extraction, numerous plant GIPCs have been characterized by mass spectrometry. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) full scan analysis of negative ions provides a quick overview of GIPC distribution. Clear differences were observed for the two plant models studied: six GIPC series bearing from two to seven saccharide units were detected in tobacco BY-2 cell extracts, whereas GIPCs extracted from A. thaliana cell cultures and leaves were less diverse, with a dominance of species containing only two saccharide units. The number of GIPC species was around 50 in A. thaliana and 120 in tobacco BY-2 cells. MALDI-MS/MS spectra gave access to detailed structural information relative to the ceramide moiety, the polar head, as well as the number and types of saccharide units. Once released from GIPCs, fatty acid chains and long-chain bases were analyzed by GC/MS to verify that all GIPC series were taken into account by the MALDI-MS/MS approach. ESI-MS/MS provided complementary information for the identification of isobaric species and fatty acid chains. Such a methodology, mostly relying on MALDI-MS/MS, should open new avenues to determine structure-function relationships between glycosphingolipids and membrane organization.

  17. An estrogen receptor model to describe the regulation of prolactin synthesis by antiestrogens in vitro.

    PubMed

    Lieberman, M E; Gorski, J; Jordan, V C

    1983-04-25

    A hypothetical model of the ligand interaction with the estrogen receptor binding site has been developed to describe the structural features necessary to initiate or to inhibit prolactin synthesis in vitro. The biological potency of the binding ligands is directly related to their relative binding affinity (RBA) for the estrogen receptor. The relative potencies of antiestrogens to inhibit estradiol-stimulated prolactin synthesis was trans-monohydroxytamoxifen identical to cis-monohydroxytamoxifen identical to tamoxifen, consistent with their RBAs for uterine estrogen receptor. Similarly the relative potency of estrogens to stimulate prolactin synthesis was diethylstilbestrol identical to estradiol greater than ICI 77,949 greater than ICI 47,699 identical to zuclomiphene, consistent with their RBAs. The compound LY126412 (trioxifene without the aminoethoxy side chain) did not interact with the estrogen receptor at the concentrations tested (10(-8)--10(-6) M) or exhibit estrogenic or antiestrogenic properties using the prolactin synthesis assay. Overall, the ligand-receptor model stresses the structural requirement for high affinity binding and the critical positioning of the alkylamino-ethoxy side chain in space (in relation to the ligand-binding site on the estrogen receptor) to prevent prolactin synthesis.

  18. ChIP-seq reveals the global regulator AlgR mediating cyclic di-GMP synthesis in Pseudomonas aeruginosa

    PubMed Central

    Kong, Weina; Zhao, Jingru; Kang, Huaping; Zhu, Miao; Zhou, Tianhong; Deng, Xin; Liang, Haihua

    2015-01-01

    AlgR is a key transcriptional regulator required for the expression of multiple virulence factors, including type IV pili and alginate in Pseudomonas aeruginosa. However, the regulon and molecular regulatory mechanism of AlgR have yet to be fully elucidated. Here, among 157 loci that were identified by a ChIP-seq assay, we characterized a gene, mucR, which encodes an enzyme that synthesizes the intracellular second messenger cyclic diguanylate (c-di-GMP). A ΔalgR strain produced lesser biofilm than did the wild-type strain, which is consistent with a phenotype controlled by c-di-GMP. AlgR positively regulates mucR via direct binding to its promoter. A ΔalgRΔmucR double mutant produced lesser biofilm than did the single ΔalgR mutant, demonstrating that c-di-GMP is a positive regulator of biofilm formation. AlgR controls the levels of c-di-GMP synthesis via direct regulation of mucR. In addition, the cognate sensor of AlgR, FimS/AlgZ, also plays an important role in P. aeruginosa virulence. Taken together, this study provides new insights into the AlgR regulon and reveals the involvement of c-di-GMP in the mechanism underlying AlgR regulation. PMID:26206672

  19. Dysregulations in circulating sphingolipids associate with disease activity indices in female patients with systemic lupus erythematosus: a cross-sectional study.

    PubMed

    Checa, A; Idborg, H; Zandian, A; Sar, D Garcia; Surowiec, I; Trygg, J; Svenungsson, E; Jakobsson, P-J; Nilsson, P; Gunnarsson, I; Wheelock, C E

    2017-01-01

    Objective The objective of this study was to investigate the association of clinical and renal disease activity with circulating sphingolipids in patients with systemic lupus erythematosus. Methods We used liquid chromatography tandem mass spectrometry to measure the levels of 27 sphingolipids in plasma from 107 female systemic lupus erythematosus patients and 23 controls selected using a design of experiment approach. We investigated the associations between sphingolipids and two disease activity indices, the Systemic Lupus Activity Measurement and the Systemic Lupus Erythematosus Disease Activity Index. Damage was scored according to the Systemic Lupus International Collaborating Clinics damage index. Renal activity was evaluated with the British Island Lupus Activity Group index. The effects of immunosuppressive treatment on sphingolipid levels were evaluated before and after treatment in 22 female systemic lupus erythematosus patients with active disease. Results Circulating sphingolipids from the ceramide and hexosylceramide families were increased, and sphingoid bases were decreased, in systemic lupus erythematosus patients compared to controls. The ratio of C16:0-ceramide to sphingosine-1-phosphate was the best discriminator between patients and controls, with an area under the receiver-operating curve of 0.77. The C16:0-ceramide to sphingosine-1-phosphate ratio was associated with ongoing disease activity according to the Systemic Lupus Activity Measurement and the Systemic Lupus Erythematosus Disease Activity Index, but not with accumulated damage according to the Systemic Lupus International Collaborating Clinics Damage Index. Levels of C16:0- and C24:1-hexosylceramides were able to discriminate patients with current versus inactive/no renal involvement. All dysregulated sphingolipids were normalized after immunosuppressive treatment. Conclusion We provide evidence that sphingolipids are dysregulated in systemic lupus erythematosus and associated with

  20. Metabolic Transition of Milk Lactose Synthesis and Up-regulation by AKT1 in Sows from Late Pregnancy to Lactation.

    PubMed

    Chen, Fang; Chen, Baoliang; Guan, Wutai; Chen, Jun; Lv, Yantao; Qiao, Hanzhen; Wang, Chaoxian; Zhang, Yinzhi

    2017-03-01

    Lactose plays a crucial role in controlling milk volume by inducing water toward into the mammary secretory vesicles from the mammary epithelial cell cytoplasm, thereby maintaining osmolality. In current study, we determined the expression of several lactose synthesis related genes, including glucose transporters (glucose transporter 1, glucose transporter 8, sodium-glucose cotransporter 1, sodium-glucose cotransporter 3, and sodium-glucose cotransporter 5), lactose synthases (α-lactalbumin and β1,4-galactosyltransferase), and hexokinases (hexokinase-1 and hexokinase-2) in sow mammary gland tissue at day 17 before delivery, on the 1st day of lactation and at peak lactation. The data showed that glucose transporter 1 was the dominant glucose transporter within sow mammary gland and that expression of each glucose transporter 1, sodium-glucose cotransporter 1, hexokinase-1, hexokinase-2, α-lactalbumin, and β1,4-galactosyltransferase were increased (p < 0.05) when the sows transited from late pregnancy to peak lactation. AKT1 over-expressed mammary epithelial cells were then constructed, and the results indicated that AKT1 increases (p < 0.01) the expression of hexokinase-1 and glucose transporter 1. In summary, lactose synthesis was significantly elevated with the increase of milk production and AKT1 could positively regulate lactose synthesis.

  1. Stress-induced cytokinin synthesis increases drought tolerance through the coordinated regulation of carbon and nitrogen assimilation in rice.

    PubMed

    Reguera, Maria; Peleg, Zvi; Abdel-Tawab, Yasser M; Tumimbang, Ellen B; Delatorre, Carla A; Blumwald, Eduardo

    2013-12-01

    The effects of water deficit on carbon and nitrogen metabolism were investigated in flag leaves of wild-type and transgenic rice (Oryza sativa japonica 'Kitaake') plants expressing ISOPENTENYLTRANSFERASE (IPT; encoding the enzyme that mediates the rate-limiting step in cytokinin synthesis) under the control of P(SARK), a maturation- and stress-induced promoter. While the wild-type plants displayed inhibition of photosynthesis and nitrogen assimilation during water stress, neither carbon nor nitrogen assimilation was affected by stress in the transgenic P(SARK)::IPT plants. In the transgenic plants, photosynthesis was maintained at control levels during stress and the flag leaf showed increased sucrose (Suc) phosphate synthase activity and reduced Suc synthase and invertase activities, leading to increased Suc contents. The sustained carbon assimilation in the transgenic P(SARK)::IPT plants was well correlated with enhanced nitrate content, higher nitrate reductase activity, and sustained ammonium contents, indicating that the stress-induced cytokinin synthesis in the transgenic plants played a role in maintaining nitrate acquisition. Protein contents decreased and free amino acids increased in wild-type plants during stress, while protein content was preserved in the transgenic plants. Our results indicate that the stress-induced cytokinin synthesis in the transgenic plants promoted sink strengthening through a cytokinin-dependent coordinated regulation of carbon and nitrogen metabolism that facilitates an enhanced tolerance of the transgenic plants to water deficit.

  2. Fas-induced programmed cell death is mediated by a Ras-regulated O2- synthesis.

    PubMed Central

    Gulbins, E; Brenner, B; Schlottmann, K; Welsch, J; Heinle, H; Koppenhoefer, U; Linderkamp, O; Coggeshall, K M; Lang, F

    1996-01-01

    Fas induces apoptosis in lymphocytes via a poorly defined intracellular signalling cascade. Previously, we have demonstrated the involvement and significance of a signalling cascade from the Fas receptor via sphingomyelinases and ceramide to Ras in Fas-induced apoptosis. Here we demonstrate rapid and transient synthesis of reactive oxygen intermediates (ROI) via activation of Ras after Fas. Genetic inhibition of Ras by transfection of transdominant inhibitory N17Ras blocked Fas-mediated ROI synthesis and programmed cell death. Likewise, the antioxidants N-acetyl-cysteine and N-t-butyl-phenylnitrone abolished Fas-induced cell death, pointing to an important role for Ras-triggered ROI synthesis in Fas-mediated programmed cell death. Images Figure 1 Figure 3 PMID:8943716

  3. NAD+-dependent Deacetylase SIRT3 Regulates Mitochondrial Protein Synthesis by Deacetylation of the Ribosomal Protein MRPL10*

    PubMed Central

    Yang, Yongjie; Cimen, Huseyin; Han, Min-Joon; Shi, Tong; Deng, Jian-Hong; Koc, Hasan; Palacios, Orsolya M.; Montier, Laura; Bai, Yidong; Tong, Qiang; Koc, Emine C.

    2010-01-01

    A member of the sirtuin family of NAD+-dependent deacetylases, SIRT3, is located in mammalian mitochondria and is important for regulation of mitochondrial metabolism, cell survival, and longevity. In this study, MRPL10 (mitochondrial ribosomal protein L10) was identified as the major acetylated protein in the mitochondrial ribosome. Ribosome-associated SIRT3 was found to be responsible for deacetylation of MRPL10 in an NAD+-dependent manner. We mapped the acetylated Lys residues by tandem mass spectrometry and determined the role of these residues in acetylation of MRPL10 by site-directed mutagenesis. Furthermore, we observed that the increased acetylation of MRPL10 led to an increase in translational activity of mitochondrial ribosomes in Sirt3−/− mice. In a similar manner, ectopic expression and knockdown of SIRT3 in C2C12 cells resulted in the suppression and enhancement of mitochondrial protein synthesis, respectively. Our findings constitute the first evidence for the regulation of mitochondrial protein synthesis by the reversible acetylation of the mitochondrial ribosome and characterize MRPL10 as a novel substrate of the NAD+-dependent deacetylase, SIRT3. PMID:20042612

  4. Mechanisms of crosstalk between endocrine systems: regulation of sex steroid hormone synthesis and action by thyroid hormones.

    PubMed

    Duarte-Guterman, Paula; Navarro-Martín, Laia; Trudeau, Vance L

    2014-07-01

    Thyroid hormones (THs) are well-known regulators of development and metabolism in vertebrates. There is increasing evidence that THs are also involved in gonadal differentiation and reproductive function. Changes in TH status affect sex ratios in developing fish and frogs and reproduction (e.g., fertility), hormone levels, and gonad morphology in adults of species of different vertebrates. In this review, we have summarized and compared the evidence for cross-talk between the steroid hormone and thyroid axes and present a comparative model. We gave special attention to TH regulation of sex steroid synthesis and action in both the brain and gonad, since these are important for gonad development and brain sexual differentiation and have been studied in many species. We also reviewed research showing that there is a TH system, including receptors and enzymes, in the brains and gonads in developing and adult vertebrates. Our analysis shows that THs influences sex steroid hormone synthesis in vertebrates, ranging from fish to pigs. This concept of crosstalk and conserved hormone interaction has implications for our understanding of the role of THs in reproduction, and how these processes may be dysregulated by environmental endocrine disruptors.

  5. The PCNA-associated protein PARI negatively regulates homologous recombination via the inhibition of DNA repair synthesis.

    PubMed

    Burkovics, Peter; Dome, Lili; Juhasz, Szilvia; Altmannova, Veronika; Sebesta, Marek; Pacesa, Martin; Fugger, Kasper; Sorensen, Claus Storgaard; Lee, Marietta Y W T; Haracska, Lajos; Krejci, Lumir

    2016-04-20

    Successful and accurate completion of the replication of damage-containing DNA requires mainly recombination and RAD18-dependent DNA damage tolerance pathways. RAD18 governs at least two distinct mechanisms: translesion synthesis (TLS) and template switching (TS)-dependent pathways. Whereas TS is mainly error-free, TLS can work in an error-prone manner and, as such, the regulation of these pathways requires tight control to prevent DNA errors and potentially oncogenic transformation and tumorigenesis. In humans, the PCNA-associated recombination inhibitor (PARI) protein has recently been shown to inhibit homologous recombination (HR) events. Here, we describe a biochemical mechanism in which PARI functions as an HR regulator after replication fork stalling and during double-strand break repair. In our reconstituted biochemical system, we show that PARI inhibits DNA repair synthesis during recombination events in a PCNA interaction-dependent way but independently of its UvrD-like helicase domain. In accordance, we demonstrate that PARI inhibits HR in vivo, and its knockdown suppresses the UV sensitivity of RAD18-depleted cells. Our data reveal a novel human regulatory mechanism that limits the extent of HR and represents a new potential target for anticancer therapy.

  6. Coordinated regulation of protein synthesis and degradation by mTORC1.

    PubMed

    Zhang, Yinan; Nicholatos, Justin; Dreier, John R; Ricoult, Stéphane J H; Widenmaier, Scott B; Hotamisligil, Gökhan S; Kwiatkowski, David J; Manning, Brendan D

    2014-09-18

    Eukaryotic cells coordinately control anabolic and catabolic processes to maintain cell and tissue homeostasis. Mechanistic target of rapamycin complex 1 (mTORC1) promotes nutrient-consuming anabolic processes, such as protein synthesis. Here we show that as well as increasing protein synthesis, mTORC1 activation in mouse and human cells also promotes an increased capacity for protein degradation. Cells with activated mTORC1 exhibited elevated levels of intact and active proteasomes through a global increase in the expression of genes encoding proteasome subunits. The increase in proteasome gene expression, cellular proteasome content, and rates of protein turnover downstream of mTORC1 were all dependent on induction of the transcription factor nuclear factor erythroid-derived 2-related factor 1 (NRF1; also known as NFE2L1). Genetic activation of mTORC1 through loss of the tuberous sclerosis complex tumour suppressors, TSC1 or TSC2, or physiological activation of mTORC1 in response to growth factors or feeding resulted in increased NRF1 expression in cells and tissues. We find that this NRF1-dependent elevation in proteasome levels serves to increase the intracellular pool of amino acids, which thereby influences rates of new protein synthesis. Therefore, mTORC1 signalling increases the efficiency of proteasome-mediated protein degradation for both quality control and as a mechanism to supply substrate for sustained protein synthesis.

  7. Inhibition of ceramide synthesis ameliorates glucocorticoid-, saturated-fat-, and obesity-induced insulin resistance.

    PubMed

    Holland, William L; Brozinick, Joseph T; Wang, Li-Ping; Hawkins, Eric D; Sargent, Katherine M; Liu, Yanqi; Narra, Krishna; Hoehn, Kyle L; Knotts, Trina A; Siesky, Angela; Nelson, Don H; Karathanasis, Sotirios K; Fontenot, Greg K; Birnbaum, Morris J; Summers, Scott A

    2007-03-01

    Insulin resistance occurs in 20%-25% of the human population, and the condition is a chief component of type 2 diabetes mellitus and a risk factor for cardiovascular disease and certain forms of cancer. Herein, we demonstrate that the sphingolipid ceramide is a common molecular intermediate linking several different pathological metabolic stresses (i.e., glucocorticoids and saturated fats, but not unsaturated fats) to the induction of insulin resistance. Moreover, inhibition of ceramide synthesis markedly improves glucose tolerance and prevents the onset of frank diabetes in obese rodents. Collectively, these data have two important implications. First, they indicate that different fatty acids induce insulin resistance by distinct mechanisms discerned by their reliance on sphingolipid synthesis. Second, they identify enzymes required for ceramide synthesis as therapeutic targets for combating insulin resistance caused by nutrient excess or glucocorticoid therapy.

  8. Lipid phosphate phosphohydrolase-1 degrades exogenous glycerolipid and sphingolipid phosphate esters.

    PubMed Central

    Jasinska, R; Zhang, Q X; Pilquil, C; Singh, I; Xu, J; Dewald, J; Dillon, D A; Berthiaume, L G; Carman, G M; Waggoner, D W; Brindley, D N

    1999-01-01

    Lipid phosphate phosphohydrolase (LPP)-1 cDNA was cloned from a rat liver cDNA library. It codes for a 32-kDa protein that shares 87 and 82% amino acid sequence identities with putative products of murine and human LPP-1 cDNAs, respectively. Membrane fractions of rat2 fibroblasts that stably expressed mouse or rat LPP-1 exhibited 3.1-3. 6-fold higher specific activities for phosphatidate dephosphorylation compared with vector controls. Increases in the dephosphorylation of lysophosphatidate, ceramide 1-phosphate, sphingosine 1-phosphate and diacylglycerol pyrophosphate were similar to those for phosphatidate. Rat2 fibroblasts expressing mouse LPP-1 cDNA showed 1.6-2.3-fold increases in the hydrolysis of exogenous lysophosphatidate, phosphatidate and ceramide 1-phosphate compared with vector control cells. Recombinant LPP-1 was located partially in plasma membranes with its C-terminus on the cytosolic surface. Lysophosphatidate dephosphorylation was inhibited by extracellular Ca2+ and this inhibition was diminished by extracellular Mg2+. Changing intracellular Ca2+ concentrations did not alter exogenous lysophosphatidate dephosphorylation significantly. Permeabilized fibroblasts showed relatively little latency for the dephosphorylation of exogenous lysophosphatidate. LPP-1 expression decreased the activation of mitogen-activated protein kinase and DNA synthesis by exogenous lysophosphatidate. The product of LPP-1 cDNA is concluded to act partly to degrade exogenous lysophosphatidate and thereby regulate its effects on cell signalling. PMID:10359651

  9. Glucocorticoids regulate surfactant protein synthesis in a pulmonary adenocarcinoma cell line

    SciTech Connect

    O'Reilly, M.A.; Gazdar, A.F.; Clark, J.C.; Pilot-Matias, T.J.; Wert, S.E.; Hull, W.M.; Whitsett, J.A. )

    1989-12-01

    Synthesis of pulmonary surfactant proteins SP-A, SP-B, and SP-C was demonstrated in a cell line derived from a human adenocarcinoma of the lung. The cells contained numerous lamellar inclusion bodies and formed organized groups of cells containing well-developed junctional complexes and apical microvillous membranes. Synthesis of SP-A was detected in the cells by enzyme-linked immunoabsorbent assay and by immunoprecipitation of (35S)methionine-labeled protein. SP-A was identified as an Mr 31,000-36,000 polypeptide containing asparagine-linked carbohydrate. Northern blot analysis detected SP-A mRNA of 2.2 kb. Dexamethasone (1-10 nM) enhanced the relative abundance of SP-A mRNA. Despite stimulation of SP-A mRNA, intracellular SP-A content was unaltered or inhibited by dexamethasone. SP-B and SP-C mRNAs and synthesis of the SP-B and SP-C precursors were markedly induced by dexamethasone. ProSP-B was synthesized and secreted primarily as an Mr 42,000-46,000 polypeptide. Proteolysis of the proSP-B resulted in the generation of endoglycosidase F-sensitive Mr = 19,000-21,000 and 25,000-27,000 peptides, which were detected both intra- and extracellularly. SP-C proprotein of Mr = 22,000 and smaller SP-C fragments were detected intracellularly but were not detected in the media. Mature forms of SP-B (Mr = 8,000) and SP-C (Mr = 4,000) were not detected. Glucocorticoids directly enhance the relative synthesis and mRNA of the surfactant proteins SP-A, SP-B, and SP-C. Discrepancies among SP-A mRNA, its de novo synthesis, and cell content suggest that glucocorticoid may alter both pre- and posttranslational factors modulating SP-A expression.

  10. Delineating Amyloid Plaque Associated Neuronal Sphingolipids in Transgenic Alzheimer’s Disease Mice (tgArcSwe) Using MALDI Imaging Mass Spectrometry

    PubMed Central

    2016-01-01

    The major pathological hallmarks of Alzheimer’s disease (AD) are the progressive aggregation and accumulation of beta-amyloid (Aβ) and hyperphosphorylated tau protein into neurotoxic deposits. Aβ aggregation has been suggested as the critical early inducer, driving the disease progression. However, the factors that promote neurotoxic Aβ aggregation remain elusive. Imaging mass spectrometry (IMS) is a powerful technique to comprehensively elucidate the spatial distribution patterns of lipids, peptides, and proteins in biological tissue sections. In the present study, matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS)-based imaging was used on transgenic Alzheimer’s disease mouse (tgArcSwe) brain tissue to investigate the sphingolipid microenvironment of individual Aβ plaques and elucidate plaque-associated sphingolipid alterations. Multivariate data analysis was used to interrogate the IMS data for identifying pathologically relevant, anatomical features based on their lipid chemical profile. This approach revealed sphingolipid species that distinctly located to cortical and hippocampal deposits, whose Aβ identity was further verified using fluorescent amyloid staining and immunohistochemistry. Subsequent multivariate statistical analysis of the spectral data revealed significant localization of gangliosides and ceramides species to Aβ positive plaques, which was accompanied by distinct local reduction of sulfatides. These plaque-associated changes in sphingolipid levels implicate a functional role of sphingolipid metabolism in Aβ plaque pathology and AD pathogenesis. Taken together, the presented data highlight the potential of imaging mass spectrometry as a powerful approach for probing Aβ plaque-associated lipid changes underlying AD pathology. PMID:27984697

  11. cAMP dependent and independent regulation of thyroglobulin synthesis by two clones of the OVNIS 6H thyroid cell line.

    PubMed

    Aouani, A; Hovsépian, S; Fayet, G

    1987-07-01

    The hormonal regulation of thyroglobulin synthesis has been studied using two independent clones of the OVNIS 6H cell line. Insulin, hydrocortisone and TSH were able to stimulate thyroglobulin synthesis, whereas transferrin, somatostatin and glycyl-histidyl-lysine were without effect. Insulin stimulated thyroglobulin synthesis without affecting cAMP production. Hydrocortisone, when combined with insulin was a stimulator too; this stimulation was not accompanied by an increase in cAMP. TSH alone was unable to stimulate either cAMP or thyroglobulin synthesis. The stimulatory effect of TSH on thyroglobulin synthesis took place only when combined with insulin or insulin plus hydrocortisone, and was mediated by cAMP. Consequently, insulin and hydrocortisone stimulated thyroglobulin synthesis by cAMP-independent mechanisms, whereas TSH acted via the cAMP system. Forskolin mimicked TSH effects on cAMP and thyroglobulin synthesis. Calf serum inhibited cAMP and thyroglobulin production. Optimal cAMP and thyroglobulin synthesis as well as TSH responsiveness were obtained in serum-free medium supplemented with 5 micrograms/ml insulin, 100 nM hydrocortisone and 1 mU/ml TSH.

  12. Regulation of type 1 fimbriae synthesis and biofilm formation by the transcriptional regulator LrhA of Escherichia coli.

    PubMed

    Blumer, Caroline; Kleefeld, Alexandra; Lehnen, Daniela; Heintz, Margit; Dobrindt, Ulrich; Nagy, Gábor; Michaelis, Kai; Emödy, Levente; Polen, Tino; Rachel, Reinhard; Wendisch, Volker F; Unden, Gottfried

    2005-10-01

    Type 1 fimbriae of Escherichia coli facilitate attachment to the host mucosa and promote biofilm formation on abiotic surfaces. The transcriptional regulator LrhA, which is known as a repressor of flagellar, motility and chemotaxis genes, regulates biofilm formation and expression of type 1 fimbriae. Whole-genome expression profiling revealed that inactivation of lrhA results in an increased expression of structural components of type 1 fimbriae. In vitro, LrhA bound to the promoter regions of the two fim recombinases (FimB and FimE) that catalyse the inversion of the fimA promoter, and to the invertible element itself. Translational lacZ fusions with these genes and quantification of fimE transcript levels by real-time PCR showed that LrhA influences type 1 fimbrial phase variation, primarily via activation of FimE, which is required for the ON-to-OFF transition of the fim switch. Enhanced type 1 fimbrial expression as a result of lrhA disruption was confirmed by mannose-sensitive agglutination of yeast cells. Biofilm formation was stimulated by lrhA inactivation and completely suppressed upon LrhA overproduction. The effects of LrhA on biofilm formation were exerted via the changed levels of surface molecules, most probably both flagella and type 1 fimbriae. Together, the data show a role for LrhA as a repressor of type 1 fimbrial expression, and thus as a regulator of the initial stages of biofilm development and, presumably, bacterial adherence to epithelial host cells also.

  13. Short-term Mg deficiency upregulates protein kinase C isoforms in cardiovascular tissues and cells; relation to NF-kB, cytokines, ceramide salvage sphingolipid pathway and PKC-zeta: hypothesis and review.

    PubMed

    Altura, Burton M; Shah, Nilank C; Shah, Gatha J; Zhang, Aimin; Li, Wenyan; Zheng, Tao; Perez-Albela, Jose Luis; Altura, Bella T

    2014-01-01

    Numerous recent,epidemiological studies reveal that Western populations are growing more and more deficient in daily Mg intake which have been linked to etiology of cardiovascular (CV) diseases. A growing body of evidence suggests that a major missing link to this dilemma may reside within the sphingolipid-ceramide pathways. For the past 25 years , our labs have been focusing on these pathways in Mg-deficient mammals. The objective of this paper is two-fold: 1) to test various hypotheses and 2) to review the current status of the field and how protein kinase C isoforms may be pivotal to solving some of the CV attributes of Mg deficiency. Below, we test the hypotheses that: 1) short-term dietary deficiency of magnesium (MgD) would result in the upregulation of protein kinase C (PKC) isoforms in left ventricular (LV) and aortic smooth muscle (ASM) and serum; 2) MgD would result in a release of select cytokines and an upregulation of NF-kB in LV and ASM, and in primary cultured aortic smooth muscle cells (PCASMC); 3) MgD would result in an activation of the sphingolipid salvage pathway in LV and ASM, and in PCASMC; 4) MgD would result in a synthesis of sphingosine, but not sphinganine, in PCASMC which could be inhibited by fumonisin B1 (FB) an inhibitor of ceramide synthase (CS), but not scyphostatin an inhibitor of neutral sphingomyelinase (N-SMase); 5) incubation of PCASMC (in low Mg(2+)) with the PKC-mimic PMA would result in release and synthesis of NF-kB, cytokines, and ceramide but not sphingosine. The new data indicate that short-term MgD (10% normal dietary intake) result in an upregulation of all three classes of PKC isoforms in LV, aortic muscle and in serum coupled to the upregulation of ceramide, NF-kB activation, and cytokines. High degrees of linear correlation were found to exist between upregulation of PKC isoforms, p65 and cytokine release, suggesting cross-talk between these molecules and molecular pathways. Our experiments with PCASMCs demonstrated

  14. Steroid synthesis by Taenia crassiceps WFU cysticerci is regulated by enzyme inhibitors.

    PubMed

    Aceves-Ramos, A; Valdez, R A; Gaona, B; Willms, K; Romano, M C

    2013-07-01

    Cysticerci and tapeworms from Taenia crassiceps WFU, ORF and Taenia solium synthesize sex-steroid hormones in vitro. Corticosteroids increase the 17β-estradiol synthesis by T. crassiceps cysticerci. T. crassiceps WFU cysticerci synthesize corticosteroids, mainly 11-deoxycorticosterone (DOC). The aim of this work was to investigate whether classical steroidogenic inhibitors modify the capacity of T. crassiceps WFU cysticerci to synthesize corticosteroids and sex steroid hormones. For this purpose, T. crassiceps WFU cysticerci were obtained from the abdominal cavity of mice, pre-cultured for 24h in DMEM+antibiotics/antimycotics and cultured in the presence of tritiated progesterone ((3)H-P4), androstendione ((3)H-A4), or dehydroepiandrosterone ((3)H-DHEA) plus different doses of the corresponding inhibitors, for different periods. Blanks with the culture media adding the tritiated precursors were simultaneously incubated. At the end of the incubation period, parasites were separated and media extracted with ether. The resulting steroids were separated by thin layer chromatography (TLC). Data were expressed as percent transformation of the tritiated precursors. Results showed that after 2h of exposure of the cysticerci to 100 μM formestane, the (3)H-17β-estradiol synthesis from tritiated androstenedione was significantly inhibited. The incubation of cysticerci in the presence of (3)H-DHEA and danazol (100 nM) resulted in (3)H-androstenediol accumulation and a significant reduction of the 17β-estradiol synthesis. The cysticerci (3)H-DOC synthesis was significantly inhibited when the parasites were cultured in the presence of different ketoconazole dosis. The drug treatments did not affect parasite's viability. The results of this study showed that corticosteroid and sex steroid synthesis in T. crassiceps WFU cysticerci can be modified by steroidogenic enzyme inhibitors. As was shown previously by our laboratory and others, parasite survival and development depends

  15. Dynamics in enzymatic protein complexes offer a novel principle for the regulation of melatonin synthesis in the human pineal gland.

    PubMed

    Maronde, Erik; Saade, Anastasia; Ackermann, Katrin; Goubran-Botros, Hany; Pagan, Cecile; Bux, Roman; Bourgeron, Thomas; Dehghani, Faramarz; Stehle, Jörg H

    2011-08-01

    Time of day is communicated to the body through rhythmic cues, including pineal gland melatonin synthesis, which is restricted to nighttime. Whereas in most rodents transcriptional regulation of the arylalkylamine N-acetyltransferase (Aanat) gene is essential for rhythmic melatonin synthesis, investigations into nonrodent mammalian species have shown post-transcriptional regulation to be of central importance, with molecular mechanisms still elusive. Therefore, human pineal tissues, taken from routine autopsies were allocated to four time-of-death groups (night/dawn/day/dusk) and analyzed for daytime-dependent changes in phosphorylated AANAT (p31T-AANAT) and in acetyl-serotonin-methyltransferase (ASMT) expression and activity. Protein content, intracellular localization, and colocalization of p31T-AANAT and ASMT were assessed, using immunoblotting, immunofluorescence, and immunoprecipitation techniques. Fresh sheep pineal gland preparations were used for comparative purposes. The amount of p31T-AANAT and ASMT proteins as well as their intracellular localization showed no diurnal variation in autoptic human and fresh sheep pineal glands. Moreover, in human and sheep pineal extracts, AANAT could not be dephosphorylated, which was at variance to data derived from rat pineal extracts. P31T-AANAT and ASMT were often found to colocalize in cellular rod-like structures that were also partly immunoreactive for the pinealocyte process-specific marker S-antigen (arrestin) in both, human and sheep pinealocytes. Protein-protein interaction studies with p31T-AANAT, ASMT, and S-antigen demonstrated a direct association and formation of robust complexes, involving also 14-3-3. This work provides evidence for a regulation principle for AANAT activity in the human pineal gland, which may not be based on a p31T-AANAT phosphorylation/dephosphorylation switch, as described for other mammalian species.

  16. Sphingolipid Metabolites Modulate Dielectric Characteristics of Cells in a Mouse Ovarian Cancer Progression Model

    PubMed Central

    Salmanzadeh, Alireza; Elvington, Elizabeth S.; Roberts, Paul C.

    2013-01-01

    Currently, conventional cancer treatment regimens often rely upon highly toxic chemotherapeutics or target oncogenes that are variably expressed within the heterogeneous cell population of tumors. These challenges highlight the need for novel treatment strategies that 1) are non-toxic yet able to at least partially reverse the aggressive phenotype of the disease to a benign or very slow-growing state, and 2) act on the cells independently of variably expressed biomarkers. Using a label-independent rapid microfluidic cell manipulation strategy known as contactless dielectrophoresis (cDEP), we investigated the effect of non-toxic concentrations of two bioactive sphingolipid metabolites, sphingosine (So), with potential anti-tumor properties, and sphingosine-1-phosphate (S1P), a tumor-promoting metabolite, on the intrinsic electrical properties of early and late stages of mouse ovarian surface epithelial (MOSE) cancer cells. Previously, we demonstrated that electrical properties change as cells progress from a benign early stage to late malignant stages. Here, we demonstrate an association between So treatment and a shift in the bioelectrical characteristics of late stage MOSE (MOSE-L) cells towards a profile similar to that of benign MOSE-E cells. Particularly, the specific membrane capacitance of MOSE-L cells shifted toward that of MOSE-E cells, decreasing from 23.94±2.75 to 16.46±0.62 mF/m2 after So treatment, associated with a decrease in membrane protrusions. In contrast, S1P did not reverse the electrical properties of MOSE-L cells. This work is the first to indicate that treatment with non-toxic doses of So correlates with changes in the electrical properties and surface roughness of cells. It also demonstrates the potential of cDEP to be used as a new, rapid technique for drug efficacy studies, and eventually designing more personalized treatment regimens. PMID:23609351

  17. Annexin II-dependent actin remodelling evoked by hydrogen peroxide requires the metalloproteinase/sphingolipid pathway.

    PubMed

    Cinq-Frais, Christel; Coatrieux, Christelle; Savary, Aude; D'Angelo, Romina; Bernis, Corinne; Salvayre, Robert; Nègre-Salvayre, Anne; Augé, Nathalie

    2015-01-01

    Actin remodeling is a dynamic process associated with cell shape modification occurring during cell cycle and proliferation. Oxidative stress plays a role in actin reorganization via various systems including p38MAPK. Beside, the mitogenic response evoked by hydrogen peroxide (H2O2) in fibroblasts and smooth muscle cells (SMC) involves the metalloproteinase (MMPs)/sphingomyelinase 2 (nSMase2) signaling pathway. The aim of this work was to investigate whether this system plays a role in actin remodeling induced by H2O2. Low H2O2 dose (5µM) rapidly triggered a signaling cascade leading to nSMase2 activation, src and annexin 2 (AnxA2) phosphorylation, and actin remodeling, in fibroblasts and SMC. These events were blocked by pharmacological inhibitors of MMPs (Ro28-2653) and p38MAPK (SB203580), and were lacking in MMP2(-/-) and in nSMase2-mutant (fro) fibroblasts. Likewise, H2O2 was unable to induce actin remodeling in fro and MMP2(-/-) fibroblasts or in cells pretreated with p38MAPK, or MMP inhibitors. Finally we show that nSMase2 activation by H2O2, depends on MMP2 and p38MAPK, and is required for the src-dependent phosphorylation of AnxA2, and actin remodeling. Taken together, these findings indicate for the first time that AnxA2 phosphorylation and actin remodeling evoked by oxidative stress depend on the sphingolipid pathway, via MMP2 and p38MAPK.

  18. Annexin II-dependent actin remodelling evoked by hydrogen peroxide requires the metalloproteinase/sphingolipid pathway

    PubMed Central

    Cinq-Frais, Christel; Coatrieux, Christelle; Savary, Aude; D’Angelo, Romina; Bernis, Corinne; Salvayre, Robert; Nègre-Salvayre, Anne; Augé, Nathalie

    2014-01-01

    Actin remodeling is a dynamic process associated with cell shape modification occurring during cell cycle and proliferation. Oxidative stress plays a role in actin reorganization via various systems including p38MAPK. Beside, the mitogenic response evoked by hydrogen peroxide (H2O2) in fibroblasts and smooth muscle cells (SMC) involves the metalloproteinase (MMPs)/sphingomyelinase 2 (nSMase2) signaling pathway. The aim of this work was to investigate whether this system plays a role in actin remodeling induced by H2O2. Low H2O2 dose (5 µM) rapidly triggered a signaling cascade leading to nSMase2 activation, src and annexin 2 (AnxA2) phosphorylation, and actin remodeling, in fibroblasts and SMC. These events were blocked by pharmacological inhibitors of MMPs (Ro28-2653) and p38MAPK (SB203580), and were lacking in MMP2−/− and in nSMase2-mutant (fro) fibroblasts. Likewise, H2O2 was unable to induce actin remodeling in fro and MMP2−/− fibroblasts or in cells pretreated with p38MAPK, or MMP inhibitors. Finally we show that nSMase2 activation by H2O2, depends on MMP2 and p38MAPK, and is required for the src-dependent phosphorylation of AnxA2, and actin remodeling. Taken together, these findings indicate for the first time that AnxA2 phosphorylation and actin remodeling evoked by oxidative stress depend on the sphingolipid pathway, via MMP2 and p38MAPK. PMID:25574848

  19. MRP- and BCL-2-mediated drug resistance in human SCLC: effects of apoptotic sphingolipids in vitro.

    PubMed

    Khodadadian, M; Leroux, M E; Auzenne, E; Ghosh, S C; Farquhar, D; Evans, R; Spohn, W; Zou, Y; Klostergaard, J

    2009-10-01

    Multidrug-resistance-associated protein (MRP) and BCL-2 contribute to drug resistance expressed in SCLC. To establish whether MRP-mediated drug resistance affects sphingolipid (SL)-induced apoptosis in SCLC, we first examined the human SCLC cell line, UMCC-1, and its MRP over-expressing, drug-resistant subline, UMCC-1/VP. Despite significantly decreased sensitivity to doxorubicin (Dox) and to the etoposide, VP-16, the drug-selected line was essentially equally as sensitive to treatment with exogenous ceramide (Cer), sphingosine (Sp) or dimethyl-sphingosine (DMSP) as the parental line. Next, we observed that high BCL-2-expressing human H69 SCLC cells, that were approximately 160-fold more sensitive to Dox than their combined BCL-2 and MRP-over-expressing (H69AR) counterparts, were only approximately 5-fold more resistant to DMSP. Time-lapse fluorescence microscopy of either UMCC cell line treated with DMSP-Coumarin revealed comparable extents and kinetics of SL uptake, further ruling out MRP-mediated effects on drug uptake. DMSP potentiated the cytotoxic activity of VP-16 and Taxol, but not Dox, in drug-resistant UMCC-1/VP cells. However, this sensitization did not appear to involve DMSP-mediated effects on the function of MRP in drug export; nor did DMSP strongly shift the balance of pro-apoptotic Sps and anti-apoptotic Sp-1-Ps in these cells. We conclude that SL-induced apoptosis markedly overcomes or bypasses MRP-mediated drug resistance relevant to SCLC and may suggest a novel therapeutic approach to chemotherapy for these tumors.

  20. Mechanisms regulating the marked seasonal variation in melatonin synthesis in the European hamster pineal gland.

    PubMed

    Garidou, Marie-Laure; Vivien-Roels, Berthe; Pevet, Paul; Miguez, Jesus; Simonneaux, Valerie

    2003-04-01

    Like many wild species, the European hamster (Cricetus cricetus) adapts to the marked seasonal changes in its environment, namely by hibernation and inhibition of sexual activity in winter. These annual functions are driven by the variation in the environmental factors (light, temperature) that are transmitted to the body through large variations in the duration and amplitude of the nocturnal melatonin rhythm. Here we report that the seasonal variation in melatonin synthesis is mainly driven by arylalkylamine N-acetyltransferase gene transcription and enzyme activation. This, however, does not exclude participation of hydroxyindole-O-methyltransferase, which may relay environmental temperature information. The in vivo experiments show that norepinephrine stimulates melatonin synthesis, this effect being gated at night. The possibility that the variation in pineal metabolism depends on a seasonal change in the suprachiasmatic nuclei clock circadian activity that is transmitted by norepinephrine is discussed.

  1. Base J glucosyltransferase does not regulate the sequence specificity of J synthesis in trypanosomatid telomeric DNA.

    PubMed

    Bullard, Whitney; Cliffe, Laura; Wang, Pengcheng; Wang, Yinsheng; Sabatini, Robert

    2015-12-01

    Telomeric DNA of trypanosomatids possesses a modified thymine base, called base J, that is synthesized in a two-step process; the base is hydroxylated by a thymidine hydroxylase forming hydroxymethyluracil (hmU) and a glucose moiety is then attached by the J-associated glucosyltransferase (JGT). To examine the importance of JGT in modifiying specific thymine in DNA, we used a Leishmania episome system to demonstrate that the telomeric repeat (GGGTTA) stimulates J synthesis in vivo while mutant telomeric sequences (GGGTTT, GGGATT, and GGGAAA) do not. Utilizing an in vitro GT assay we find that JGT can glycosylate hmU within any sequence with no significant change in Km or kcat, even mutant telomeric sequences that are unable to be J-modified in vivo. The data suggests that JGT possesses no DNA sequence specificity in vitro, lending support to the hypothesis that the specificity of base J synthesis is not at the level of the JGT reaction.

  2. The identification of translesion DNA synthesis regulators: inhibitors in the spotlight

    PubMed Central

    Bertolin, AP; Mansilla, SF; Gottifredi, V

    2015-01-01

    Over the past half-century, we have become increasingly aware of the ubiquity of DNA damage. Under the constant exposure to exogenous and endogenous genomic stress, cells must attempt to replicate damaged DNA. The encounter of replication forks with DNA lesions triggers several cellular responses, including the activation of translesion DNA synthesis (TLS), which largely depends upon specialized DNA polymerases with flexible active sites capable of accommodating bulky DNA lesions. A detrimental aspect of TLS is its intrinsic mutagenic nature, and thus the activity of the TLS polymerases must ideally be restricted to synthesis on damaged DNA templates. Despite their potential clinical importance in chemotherapy, TLS inhibitors have been difficult to identify since a direct assay designed to quantify genomic TLS events is still unavailable. Herein we discuss the methods that have been used to validate TLS inhibitors such as USP1, p21 and Spartan, highlighting research that has revealed their contribution to the control of DNA synthesis on damaged and undamaged templates. PMID:26002196

  3. Regulation of argA operon expression in Escherichia coli K-12: cell-free synthesis of beta-galactosidase under argA control.

    PubMed Central

    Kelker, N; Eckhardt, T

    1977-01-01

    Regulation of argA operon expression in Escherichia coli K-12 was studied in a cell-free, deoxyribonucleic acid-dependent, enzyme-synthesizing system. lambdaAZ-7 deoxyribonucleic acid, which carries a fusion of the lacZ structural gene to the argA operon so that beta-galactosidase synthesis is under argA regulation, was used as the template. To eliminate extraneous readthrough from lambda promoters, lambda repressor was introduced into the synthesis mixtures by preparing the S-30 component from a strain (514X5a-12-29) that carries a multicopy hybrid plasmid (pKB252) containing the lambdacI gene. Under these conditions beta-galactosidase synthesis was repressed 90% by the arginine repressor when a sufficient concentration of L-arginine was present. This repression could be overcome by escape synthesis when the lambdaAZ-7 deoxyribonucleic acid concentration in the synthesis mixtures was increased. Guanosine 3'-diphosphate-5'-diphosphate stimulated beta-galactosidase synthesis from this template. PMID:410786

  4. The regulation of glucose on milk fat synthesis is mediated by the ubiquitin-proteasome system in bovine mammary epithelial cells.

    PubMed

    Liu, Lily; Jiang, Li; Ding, Xiang-dong; Liu, Jian-feng; Zhang, Qin

    2015-09-11

    Glucose as one of the nutrition factors plays a vital role in the regulation of milk fat synthesis. Ubiquitin-proteasome system (UPS) is a vital proteolytic pathway in all eukaryotic cells through timely marking, recognizing and degrading the poly-ubiquitinated protein substrates. Previous studies indicated that UPS plays a considerable role in controlling the triglyceride (TG) synthesis. Therefore, the aim of this study is to confirm the link between high-glucose and UPS and its regulation mechanism on milk fat synthesis in BMEC (bovine mammary epithelial cells). We incubated BMEC with normal (17.5 mm/L) and high-glucose (25 mm/L) with and without proteasome inhibitor epoxomicin and found that, compared with the control (normal glucose and without proteasome inhibitor), both high-glucose concentration and proteasome inhibitor epoxomicin could increase the accumulation of TG and poly-ubiquitinated proteins, and reduce significantly three proteasome activities (chymotrypsin-like, caspase-like, and trypsin-like). In addition, high-glucose concentration combined with proteasome inhibitor further enhanced the increase of the poly-ubiquitinated protein level and the decrease of proteasome activities. Our results suggest that the regulation of high-glucose on milk fat synthesis is mediated by UPS in BMEC, and high-glucose exposure could lead to a hypersensitization of BMEC to UPS inhibition which in turn results in increased milk fat synthesis.

  5. Functional effects of a pathogenic mutation in Cereblon (CRBN) on the regulation of protein synthesis via the AMPK-mTOR cascade.

    PubMed

    Lee, Kwang Min; Yang, Seung-Joo; Choi, Ja-Hyun; Park, Chul-Seung

    2014-08-22

    Initially identified as a protein implicated in human mental deficit, cereblon (CRBN) was recently recognized as a negative regulator of adenosine monophosphate-activated protein kinase (AMPK) in vivo and in vitro. Here, we present results showing that CRBN can effectively regulate new protein synthesis through the mammalian target of rapamycin (mTOR) signaling pathway, a downstream target of AMPK. Whereas deficiency of Crbn repressed protein translation via activation of the AMPK-mTOR cascade in Crbn-knock-out mice, ectopic expression of the wild-type CRBN increased protein synthesis by inhibiting endogenous AMPK. Unlike the wild-type CRBN, a mutant CRBN found in human patients, which lacks the last 24 amino acids, failed to rescue mTOR-dependent repression of protein synthesis in Crbn-deficient mouse fibroblasts. These results provide the first evidence that Crbn can activate the protein synthesis machinery through the mTOR signaling pathway by inhibiting AMPK. In light of the fact that protein synthesis regulated by mTOR is essential for various forms of synaptic plasticity that underlie the cognitive functions of the brain, the results of this study suggest a plausible mechanism for CRBN involvement in higher brain function in humans, and they may help explain how a specific mutation in CRBN can affect the cognitive ability of patients.

  6. Down-Regulation by Resveratrol of Basic Fibroblast Growth Factor-Stimulated Osteoprotegerin Synthesis through Suppression of Akt in Osteoblasts

    PubMed Central

    Kuroyanagi, Gen; Otsuka, Takanobu; Yamamoto, Naohiro; Matsushima-Nishiwaki, Rie; Nakakami, Akira; Mizutani, Jun; Kozawa, Osamu; Tokuda, Haruhiko

    2014-01-01

    It is firmly established that resveratrol, a natural food compound abundantly found in grape skins and red wine, has beneficial properties for human health. In the present study, we investigated the effect of basic fibroblast growth factor (FGF-2) on osteoprotegerin (OPG) synthesis in osteoblast-like MC3T3-E1 cells and whether resveratrol affects the OPG synthesis. FGF-2 stimulated both the OPG release and the expression of OPG mRNA. Resveratrol significantly suppressed the FGF-2-stimulated OPG release and the mRNA levels of OPG. SRT1720, an activator of SIRT1, reduced the FGF-2-induced OPG release and the OPG mRNA expression. PD98059, an inhibitor of upstream kinase activating p44/p42 mitogen-activated protein (MAP) kinase, had little effect on the FGF-2-stimulated OPG release. On the other hand, SB203580, an inhibitor of p38 MAP kinase, SP600125, an inhibitor of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), and Akt inhibitor suppressed the OPG release induced by FGF-2. Resveratrol failed to affect the FGF-2-induced phosphorylation of p44/p42 MAP kinase, p38 MAP kinase or SAPK/JNK. The phosphorylation of Akt induced by FGF-2 was significantly suppressed by resveratrol or SRT1720. These findings strongly suggest that resveratrol down-regulates FGF-2-stimulated OPG synthesis through the suppression of the Akt pathway in osteoblasts and that the inhibitory effect of resveratrol is mediated at least in part by SIRT1 activation. PMID:25290095

  7. Amino acid availability regulates S6K1 and protein synthesis in avian insulin-insensitive QM7 myoblasts.

    PubMed

    Tesseraud, Sophie; Bigot, Karine; Taouis, Mohammed

    2003-04-10

    The regulation of S6K1 by nutritional status and insulin has been recently reported in vivo in chicken muscle despite the relative insulin resistance of this tissue as estimated by phosphatidylinositol 3-kinase (PI3-kinase) activity. The present work aimed to study the impact of amino acids on S6K1 activity in quail muscle (QM7) myoblasts. Firstly, we characterized S6K1 in QM7 cells and demonstrated the absence of insulin receptors in these cells. Secondly, we showed that amino acids in the absence of insulin induced S6K1 phosphorylation on Thr389 and concomitantly increased its enzymatic activity. Amino acid-induced S6K1 activation was inhibited by LY294002 (PI3-kinase inhibitor) and rapamycin (inhibitor of the mammalian target of rapamycin, mTOR), suggesting the involvement of an avian homolog of mTOR. The availability of individual amino acids (methionine or leucine) regulated S6K1 phosphorylation on Thr389 and QM7 protein synthesis. In conclusion, amino acids regulate S6K1 phosphorylation and activity in QM7 cells through the mTOR/PI3-kinase pathway in an insulin-independent manner.

  8. miR-375 Negatively Regulates the Synthesis and Secretion of Catecholamines by Targeting Sp1 in Rat Adrenal Medulla.

    PubMed

    Gai, Yedan; Zhang, Jinglin; Wei, Chao; Cao, Wei; Cui, Yan; Cui, Sheng

    2017-03-29

    Adrenal gland is a crucial endocrine gland, and the most important function is to synthesize and secrete catecholamines (CATs) which play crucial roles in balancing homeostasis and the responding to stress. microRNA-375 (miR-375) has been detected to highly express in the adrenal, however its role and underlying mechanism are currently unclear. Herein, our results showed that miR-375 was specifically localized to the rat adrenal medulla chromaffin cells, and miR-375 expressing level decreased, when the rats were exposed to stress. The further functional studies demonstrated that the inhibition of endogenous miR-375 induced the secretion of CATs in primary rat medulla chromaffin cells and in PC12 cells, and over-expression of miR-375 resulted in decline of the CATs secretion. Furthermore, the results showed that miR-375 negatively regulated tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DBH) and mediated adrenomedullary CATs biosynthesis. Sp1(a transcriptional activator of TH and DBH) was involved in mediating the regulation of TH and DBH as miR-375 direct target gene. These novel findings suggest that miR-375 acts as a potent negative mediator in regulating the synthesis and secretion of CATs in the adrenal medulla during the maintenance of homeostasis under stress.

  9. Ultra-high-performance liquid chromatography electrospray ionization tandem mass spectrometry for accurate analysis of glycerophospholipids and sphingolipids in drug resistance tumor cells.

    PubMed

    Li, Lin; Wang, Linlin; Shangguan, Dihua; Wei, Yanbo; Han, Juanjuan; Xiong, Shaoxiang; Zhao, Zhenwen

    2015-02-13

    Glycerophospholipids and sphingolipids are important signaling molecules which are involved in many physiological and pathological processes. Here we reported an effective method for accurate analysis of these lipids by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The methanol method was adopted for extraction of lipids due to its simplicity and high efficiency. It was found that two subclasses of sphingolipids, sulfatide (ST) and cerebroside (CB), were heat labile, so a decreased temperature in the ion source of MS might be necessary for these compounds analysis. In addition, it was found that the isobaric interferences were commonly existent, for example, the m/z of 16:0/18:1 PC containing two (13)C isotope being identical to that of 16:0/18:0 PC determined by a unit mass resolution mass spectrometer; therefore, a baseline separation of interferential species was required to maintain selectivity and accuracy of analysis. In this work, an ultra-high-performance liquid chromatography (UHPLC)-based method was developed for separation of interferential species. Moreover, in order to deal with the characteristics of different polarity and wide dynamic range of glycerophospholipids and sphingolipids in biological systems, three detecting conditions were combined together for comprehensive and rational analysis of glycerophospholipids and sphingolipids. The method was utilized to profile glycerophospholipids and sphingolipids in drug resistant tumor cells. Our results showed that many lipids were significantly changed in drug resistant tumor cells compared to paired drug sensitive tumor cells. This is a systematic report about the isobaric interferences and heat labile compounds interferences when analyzing glycerophospholipids and sphingolipids by ESI-MS/MS, which aids in ruling out one potential source of systematic error to ensure the accuracy of analysis.

  10. Neverland regulates embryonic moltings through the regulation of ecdysteroid synthesis in the water flea Daphnia magna, and may thus act as a target for chemical disruption of molting.

    PubMed

    Sumiya, Eri; Ogino, Yukiko; Toyota, Kenji; Miyakawa, Hitoshi; Miyagawa, Shinichi; Iguchi, Taisen

    2016-11-01

    Embryo development in arthropods is accompanied by a series of moltings. A cladoceran crustacean Daphnia magna molts three times before reaching first instar neonate during embryogenesis. Previous studies argued ecdysteroids might regulate D. magna embryogenesis. However, no direct evidence between innate ecdysteroids fluctuation and functions has been forthcoming. Recently, we identified genes involved in ecdysteroid synthesis called, neverland (neverland1 and neverland 2) and shade and in the ecdysteroid degradation (Cyp18a1). To understand the physiological roles of ecdysteroids in D. magna embryos, we performed expression and functional analyzes of those genes. Examining innate ecdysteroids titer during embryogenesis showed two surges of ecdysteroids titer at 41 and 61 h after oviposition. The first and second embryonic moltings occurred at each ecdysteroid surge. Expression of neverland1 and shade began to increase before the first peak in ecdysteroid. Knockdown of neverland1 or shade by RNAi technique caused defects in embryonic moltings and subsequent development. The ecdysteroids titer seemingly decreased in nvd1-knowckdown embryos. Knockdown of Cyp18a1 resulted in early embryonic lethality before the first molting. Our in situ hybridization analysis revealed that nvd1 was prominently expressed in embryonic gut epithelium suggesting the site for an initial step of ecdysteroidgenesis, a conversion of cholesterol to 7-dehydrocholesterol and possibly for ecdysone production. Taken together, de novo ecdysteroid synthesis by nvd1 in the gut epithelial cells stimulates molting, which is indispensable for D. magna embryo development. These findings identify neverland as a possible target for chemicals, including various pesticides that are known to disrupt molting, development and reproduction. Copyright © 2016 John Wiley & Sons, Ltd.

  11. SND1, a NAC domain transcription factor, is a key regulator of secondary wall synthesis in fibers of Arabidopsis.

    PubMed

    Zhong, Ruiqin; Demura, Taku; Ye, Zheng-Hua

    2006-11-01

    Secondary walls in fibers and tracheary elements constitute the most abundant biomass produced by plants. Although a number of genes involved in the biosynthesis of secondary wall components have been characterized, little is known about the molecular mechanisms underlying the coordinated expression of these genes. Here, we demonstrate that the Arabidopsis thaliana NAC (for NAM, ATAF1/2, and CUC2) domain transcription factor, SND1 (for secondary wall-associated NAC domain protein), is a key transcriptional switch regulating secondary wall synthesis in fibers. We show that SND1 is expressed specifically in interfascicular fibers and xylary fibers in stems and that dominant repression of SND1 causes a drastic reduction in the secondary wall thickening of fibers. Ectopic overexpression of SND1 results in activation of the expression of secondary wall biosynthetic genes, leading to massive deposition of secondary walls in cells that are normally nonsclerenchymatous. In addition, we have found that SND1 upregulates the expression of several transcription factors that are highly expressed in fibers during secondary wall synthesis. Together, our results reveal that SND1 is a key transcriptional activator involved in secondary wall biosynthesis in fibers.

  12. Saccharomyces cerevisiae mutant with a partial defect in the synthesis of CDP-diacylglycerol and altered regulation of phospholipid biosynthesis.

    PubMed Central

    Klig, L S; Homann, M J; Kohlwein, S D; Kelley, M J; Henry, S A; Carman, G M

    1988-01-01

    A Saccharomyces cerevisiae mutant (cdg1 mutation) was isolated on the basis of an inositol excretion phenotype and exhibited pleiotropic deficiencies in phospholipid biosynthesis. Genetic analysis of the mutant confirmed that the cdg1 mutation represents a new genetic locus and that a defect in a single gene was responsible for the Cdg1 phenotype. CDP-diacylglycerol synthase activity in mutant haploid cells was 25% of the wild-type derepressed level. Biochemical and immunoblot analyses revealed that the defect in CDP-diacylglycerol synthase activity in the cdg1 mutant was due to a reduced level of the CDP-diacylglycerol synthase Mr-56,000 subunit rather than to an alteration in the enzymological properties of the enzyme. This defect resulted in a reduced rate of CDP-diacylglycerol synthesis, an elevated phosphatidate content, and alterations in overall phospholipid synthesis. Unlike wild-type cells, CDP-diacylglycerol synthase was not regulated in response to water-soluble phospholipid precursors. The cdg1 lesion also caused constitutive expression of inositol-1-phosphate synthase and elevated phosphatidylserine synthase. Phosphatidylinositol synthase was not affected in the cdg1 mutant. Images PMID:2832385

  13. Sec14 Like PITPs Couple Lipid Metabolism with Phosphoinositide Synthesis to Regulate Golgi Functionality

    PubMed Central

    Davison, James M.; Bankaitis, Vytas A.

    2017-01-01

    An interface coordinating lipid metabolism with proteins that regulate membrane trafficking is necessary to regulate Golgi morphology and dynamics. Such an interface facilitates the membrane deformations required for vesicularization, forms platforms for protein recruitment and assembly on appropriate sites on a membrane surface and provides lipid co-factors for optimal protein activity in the proper spatio-temporally regulated manner. Importantly, Sec14 and Sec14-like proteins are a unique superfamily of proteins that sense specific aspects of lipid metabolism, employing this information to potentiate phosphoinositide production. Therefore, Sec14 and Sec14 like proteins form central conduits to integrate multiple aspects of lipid metabolism with productive phosphoinositide signaling. PMID:22374094

  14. Neural-genetic synthesis for state-space controllers based on linear quadratic regulator design for eigenstructure assignment.

    PubMed

    da Fonseca Neto, João Viana; Abreu, Ivanildo Silva; da Silva, Fábio Nogueira

    2010-04-01

    Toward the synthesis of state-space controllers, a neural-genetic model based on the linear quadratic regulator design for the eigenstructure assignment of multivariable dynamic systems is presented. The neural-genetic model represents a fusion of a genetic algorithm and a recurrent neural network (RNN) to perform the selection of the weighting matrices and the algebraic Riccati equation solution, respectively. A fourth-order electric circuit model is used to evaluate the convergence of the computational intelligence paradigms and the control design method performance. The genetic search convergence evaluation is performed in terms of the fitness function statistics and the RNN convergence, which is evaluated by landscapes of the energy and norm, as a function of the parameter deviations. The control problem solution is evaluated in the time and frequency domains by the impulse response, singular values, and modal analysis.

  15. Microfluidic Synthesis of Hybrid Nanoparticles with Controlled Lipid Layers: Understanding Flexibility-Regulated Cell-Nanoparticle Interaction.

    PubMed

    Zhang, Lu; Feng, Qiang; Wang, Jiuling; Zhang, Shuai; Ding, Baoquan; Wei, Yujie; Dong, Mingdong; Ryu, Ji-Young; Yoon, Tae-Young; Shi, Xinghua; Sun, Jiashu; Jiang, Xingyu

    2015-10-27

    The functionalized lipid shell of hybrid nanoparticles plays an important role for improving their biocompatibility and in vivo stability. Yet few efforts have been made to critically examine the shell structure of nanoparticles and its effect on cell-particle interaction. Here we develop a microfluidic chip allowing for the synthesis of structurally well-defined lipid-polymer nanoparticles of the same sizes, but covered with either lipid-monolayer-shell (MPs, monolayer nanoparticles) or lipid-bilayer-shell (BPs, bilayer nanoparticles). Atomic force microscope and atomistic simulations reveal that MPs have a lower flexibility than BPs, resulting in a more efficient cellular uptake and thus anticancer effect than BPs do. This flexibility-regulated cell-particle interaction may have important implications for designing drug nanocarriers.

  16. Ceramidase Regulates Synaptic Vesicle Exocytosis and Trafficking

    PubMed Central

    Rohrbough, Jeffrey; Rushton, Emma; Palanker, Laura; Woodruff, Elvin; Matthies, Heinrich J. G.; Acharya, Usha; Acharya, Jairaj K.; Broadie, Kendal

    2009-01-01

    A screen for Drosophila synaptic dysfunction mutants identified slug-a-bed (slab). The slab gene encodes ceramidase, a central enzyme in sphingolipid metabolism and regulation. Sphingolipids are major constituents of lipid rafts, membrane domains with roles in vesicle trafficking, and signaling pathways. Null slab mutants arrest as fully developed embryos with severely reduced movement. The SLAB protein is widely expressed in different tissues but enriched in neurons at all stages of development. Targeted neuronal expression of slab rescues mutant lethality, demonstrating the essential neuronal function of the protein. C5-ceramide applied to living preparations is rapidly accumulated at neuromuscular junction (NMJ) synapses dependent on the SLAB expression level, indicating that synaptic sphingolipid trafficking and distribution is regulated by SLAB function. Evoked synaptic currents at slab mutant NMJs are reduced by 50–70%, whereas postsynaptic glutamate-gated currents are normal, demonstrating a specific presynaptic impairment. Hypertonic saline-evoked synaptic vesicle fusion is similarly impaired by 50–70%, demonstrating a loss of readily releasable vesicles. In addition, FM1-43 dye uptake is reduced in slab mutant presynaptic terminals, indicating a smaller cycling vesicle pool. Ultrastructural analyses of mutants reveal a normal vesicle distribution clustered and docked at active zones, but fewer vesicles in reserve regions, and a twofold to threefold increased incidence of vesicles linked together and tethered at the plasma membrane. These results indicate that SLAB ceramidase function controls presynaptic terminal sphingolipid composition to regulate vesicle fusion and trafficking, and thus the strength and reliability of synaptic transmission. PMID:15356190

  17. The human testis determining factor SRY localizes in midbrain dopamine neurons and regulates multiple components of catecholamine synthesis and metabolism

    PubMed Central

    Czech, Daniel P.; Lee, Joohyung; Sim, Helena; Parish, Clare L.; Vilain, Eric; Harley, Vincent R.

    2012-01-01

    The male sex is determined by the sex determining region on the Y chromosome (SRY) transcription factor. The unexpected action of SRY in the control of voluntary movement in male rodents suggests a role in regulation of dopamine transmission and dopamine-related disorders with sex bias such as Parkinson’s disease. We investigated SRY expression in the human brain and function in vitro. SRY immunoreactivity was detected in the human male, but not female, substantia nigra pars compacta (SNc) within a sub-population of tyrosine hydroxylase (TH) positive neurons. SRY protein also co-localised with TH positive neurons in the ventral tegmental area and GAD-positive neurons in the substantia nigra pars reticulate (SNr). Retinoic acid-induced differentiation of precursor NT2 cells into dopaminergic cells (NT2N) increased expression of TH, NURR1, D2R and SRY. In the human neuroblastoma cell line, M17, SRY knockdown resulted in a reduction in TH, DDC, DBH and MAO-A expression; enzymes which control dopamine synthesis and metabolism. Conversely, SRY overexpression increased TH, DDC, DBH, D2R and MAO-A levels, which was accompanied by increased extracellular dopamine levels. A luciferase assay demonstrated that SRY activated a 4.6 kb 5′ upstream regulatory region of the human TH promoter/nigral enhancer. Combined, these results suggest that SRY may play a role as a positive regulator of catecholamine synthesis and metabolism in the human male midbrain. Given the limitations of human tissue analysis, further studies are required to provide a definitive answer on SRY expression in human brain regions. PMID:22568433

  18. Cloning of the RHO1 gene from Candida albicans and its regulation of beta-1,3-glucan synthesis.

    PubMed Central

    Kondoh, O; Tachibana, Y; Ohya, Y; Arisawa, M; Watanabe, T

    1997-01-01

    The Saccharomyces cerevisiae RHO1 gene encodes a low-molecular-weight GTPase. One of its recently identified functions is the regulation of beta-1,3-glucan synthase, which synthesizes the main component of the fungal cell wall (J. Drgonova et al., Science 272:277-279, 1996; T. Mazur and W. Baginsky, J. Biol. Chem. 271:14604-14609, 1996; and H. Qadota et al., Science 272:279-281, 1996). From the opportunistic pathogenic fungus Candida albicans, we cloned the RHO1 gene by the PCR and cross-hybridization methods. Sequence analysis revealed that the Candida RHO1 gene has a 597-nucleotide region which encodes a putative 22.0-kDa peptide. The deduced amino acid sequence predicts that Candida albicans Rho1p is 82.9% identical to Saccharomyces Rho1p and contains all the domains conserved among Rho-type GTPases from other organisms. The Candida albicans RHO1 gene could rescue a S. cerevisiae strain containing a rho1 deletion. Furthermore, recombinant Candida albicans Rho1p could reactivate the beta-1,3-glucan synthesis activities of both C. albicans and S. cerevisiae membranes in which endogenous Rho1p had been depleted by Tergitol NP-40-NaCl treatment. Candida albicans Rho1p was copurified with the beta-1,3-glucan synthase putative catalytic subunit, Candida albicans Gsc1p, by product entrapment. Candida albicans Rho1p was shown to interact directly with Candida albicans Gsc1p in a ligand overlay assay and a cross-linking study. These results indicate that Candida albicans Rho1p acts in the same manner as Saccharomyces cerevisiae Rho1p to regulate beta-1,3-glucan synthesis. PMID:9401032

  19. Nuclear Factor of κB1 Is a Key Regulator for the Transcriptional Activation of Milk Synthesis in Bovine Mammary Epithelial Cells.

    PubMed

    Huang, Xin; Zang, Yanli; Zhang, Minghui; Yuan, Xiaohan; Li, Meng; Gao, Xuejun

    2017-02-03

    The nuclear factor of κB (NFκB) family has been well known for its significant role in regulating the expression of numerous genes that control many biological processes. However, it is unclear whether NFκB could regulate milk synthesis. In this study, we identified NFκB1 as a critical regulator for milk synthesis in bovine mammary epithelial cells (BMECs). Gene function study revealed that NFκB1 modulates the expression of mammalian target of rapamycin (mTOR), sterol response element-binding protein (SREBP)-1c, and β4Gal-T2 for milk synthesis. Furthermore, chromatin immunoprecipitation assays showed that both methionine (Met) and estrogen (E) triggered NFκB1 to bind to gene promoters of mTOR, SREBP-1c, and β4Gal-T2 in BMECs. In addition, we confirmed that Met and E triggered NFκB1 expression and phosphorylation via phosphatidylinositol-3-kinase (PI3K) but not mTOR signaling pathway. Taken together, our study reveals that NFκB1 acts as a PI3K but not mTOR-dependent critical mediator for the transcriptional activation of signaling molecules regulating milk synthesis in BMECs.

  20. Identification of the Main Regulator Responsible for Synthesis of the Typical Yellow Pigment Produced by Trichoderma reesei

    PubMed Central

    Derntl, Christian; Rassinger, Alice; Srebotnik, Ewald; Mach, Robert L.

    2016-01-01

    ABSTRACT The industrially used ascomycete Trichoderma reesei secretes a typical yellow pigment during cultivation, while other Trichoderma species do not. A comparative genomic analysis suggested that a putative secondary metabolism cluster, containing two polyketide-synthase encoding genes, is responsible for the yellow pigment synthesis. This cluster is conserved in a set of rather distantly related fungi, including Acremonium chrysogenum and Penicillium chrysogenum. In an attempt to silence the cluster in T. reesei, two genes of the cluster encoding transcription factors were individually deleted. For a complete genetic proof-of-function, the genes were reinserted into the genomes of the respective deletion strains. The deletion of the first transcription factor (termed yellow pigment regulator 1 [Ypr1]) resulted in the full abolishment of the yellow pigment formation and the expression of most genes of this cluster. A comparative high-pressure liquid chromatography (HPLC) analysis of supernatants of the ypr1 deletion and its parent strain suggested the presence of several yellow compounds in T. reesei that are all derived from the same cluster. A subsequent gas chromatography/mass spectrometry analysis strongly indicated the presence of sorbicillin in the major HPLC peak. The presence of the second transcription factor, termed yellow pigment regulator 2 (Ypr2), reduces the yellow pigment formation and the expression of most cluster genes, including the gene encoding the activator Ypr1. IMPORTANCE Trichoderma reesei is used for industry-scale production of carbohydrate-active enzymes. During growth, it secretes a typical yellow pigment. This is not favorable for industrial enzyme production because it makes the downstream process more complicated and thus increases operating costs. In this study, we demonstrate which regulators influence the synthesis of the yellow pigment. Based on these data, we also provide indication as to which genes are under the control

  1. Nutritional regulation of JH synthesis: a mechanism to control reproductive maturation in mosquitoes?

    PubMed

    Noriega, Fernando G

    2004-07-01

    Juvenile hormone (JH) titers must be modulated to permit the normal progress of development and reproduction in mosquitoes. In adult female Aedes aegypti, JH levels are low at adult eclosion, elevated in sugar-fed females and low again after a blood meal. Although degradation plays a role, JH titer is fundamentally determined by the rate of biosynthesis in the corpora allata gland (CA). CA from newly eclosed females (0-1 h after emergence) exhibit a very low basal JH biosynthetic activity, Aedes-allatotropin stimulates the CA in newly emerged females to produce JH. There is a correlation between nutritional reserves at adult emergence (teneral reserves) and CA activity. JH synthesis is significantly reduced in teneral females that emerge with low nutritional reserves. Taking a blood meal results in a reduction of CA activity. The biosynthetic activity of Ae. aegypti CA is significantly inhibited by factors present in the head, as well as by Anopheles gambiae PISCF-allatostatin. Nutritional signals affect the release of allatotropin and allatostatins by the brain resulting in the activation or inhibition of JH synthesis. JH is therefore an important part of a transduction mechanism that connects changes in the nutritional status with activation of specific physiological events during reproduction.

  2. PTEN Regulates Glutamine Flux to Pyrimidine Synthesis and Sensitivity to Dihydroorotate Dehydrogenase Inhibition.

    PubMed

    Mathur, Deepti; Stratikopoulos, Elias; Ozturk, Sait; Steinbach, Nicole; Pegno, Sarah; Schoenfeld, Sarah; Yong, Raymund; Murty, Vundavalli V; Asara, John M; Cantley, Lewis C; Parsons, Ramon

    2017-04-01

    Metabolic changes induced by oncogenic drivers of cancer contribute to tumor growth and are attractive targets for cancer treatment. Here, we found that increased growth of PTEN-mutant cells was dependent on glutamine flux through the de novo pyrimidine synthesis pathway, which created sensitivity to the inhibition of dihydroorotate dehydrogenase, a rate-limiting enzyme for pyrimidine ring synthesis. S-phase PTEN-mutant cells showed increased numbers of replication forks, and inhibitors of dihydroorotate dehydrogenase led to chromosome breaks and cell death due to inadequate ATR activation and DNA damage at replication forks. Our findings indicate that enhanced glutamine flux generates vulnerability to dihydroorotate dehydrogenase inhibition, which then causes synthetic lethality in PTEN-deficient cells due to inherent defects in ATR activation. Inhibition of dihydroorotate dehydrogenase could thus be a promising therapy for patients with PTEN-mutant cancers.Significance: We have found a prospective targeted therapy for PTEN-deficient tumors, with efficacy in vitro and in vivo in tumors derived from different tissues. This is based upon the changes in glutamine metabolism, DNA replication, and DNA damage response which are consequences of inactivation of PTENCancer Discov; 7(4); 380-90. ©2017 AACR.See related article by Brown et al., p. 391This article is highlighted in the In This Issue feature, p. 339.

  3. The Ebola Virus VP30-NP Interaction Is a Regulator of Viral RNA Synthesis

    PubMed Central

    Kirchdoerfer, Robert N.; Moyer, Crystal L.; Abelson, Dafna M.; Saphire, Erica Ollmann

    2016-01-01

    Filoviruses are capable of causing deadly hemorrhagic fevers. All nonsegmented negative-sense RNA-virus nucleocapsids are composed of a nucleoprotein (NP), a phosphoprotein (VP35) and a polymerase (L). However, the VP30 RNA-synthesis co-factor is unique to the filoviruses. The assembly, structure, and function of the filovirus RNA replication complex remain unclear. Here, we have characterized the interactions of Ebola, Sudan and Marburg virus VP30 with NP using in vitro biochemistry, structural biology and cell-based mini-replicon assays. We have found that the VP30 C-terminal domain interacts with a short peptide in the C-terminal region of NP. Further, we have solved crystal structures of the VP30-NP complex for both Ebola and Marburg viruses. These structures reveal that a conserved, proline-rich NP peptide binds a shallow hydrophobic cleft on the VP30 C-terminal domain. Structure-guided Ebola virus VP30 mutants have altered affinities for the NP peptide. Correlation of these VP30-NP affinities with the activity for each of these mutants in a cell-based mini-replicon assay suggests that the VP30-NP interaction plays both essential and inhibitory roles in Ebola virus RNA synthesis. PMID:27755595

  4. Regulation of gene expression in prediapausing embryos of the silkworm, Bombyx mori: pattern of protein synthesis.

    PubMed

    Dorel, C; Coulon, M

    1988-03-01

    Specific qualitative and quantitative changes in protein synthesis occur from the fertilization to the onset of diapause in the silkworm. We have used two-dimensional gel electrophoresis to analyse the patterns of proteins synthesized in prediapausing eggs of Bombyx. This analysis has been carried out with in vivo labelled polypeptides and with proteins synthesized in vitro by RNA isolated at different stages. The oocyte contains an abundant supply of diverse mRNA which are translatable in vitro. A set of proteins with molecular weight range of 68,000 to 74,000 and isoelectric points of 5.85-5.95 (hereafter referred to as No. 30) is specific of the germ-anlage stage. Transcripts encoding the No. 30 proteins are not detectable in oocytes, and inhibition of transcription by actinomycin D indicates that No. 30 mRNA are synthesized de novo. Treating eggs at the germ-anlage stage with 4 N HCl at 46 degrees C prevents diapause and is accompanied by overproduction of No. 30 protein. The induction of No. 30 synthesis is also the main event of the heat shock response. The implications of these findings in relation to early embryonic development and prevention of diapause are discussed.

  5. Glycine as a regulator of tryptophan-dependent pigment synthesis in Malassezia furfur.

    PubMed

    Barchmann, Thorsten; Hort, Wiebke; Krämer, Hans-Joachim; Mayser, Peter

    2011-01-01

    The effects of the addition of different amino nitrogens on growth, morphology and secondary metabolism of Malassezia furfur were investigated. After primary culture on Dixon agar, M. furfur CBS 1878 was transferred into a fluid medium together with the nitrogen sources, glycine (Gly) or tryptophan (Trp), or a combination of both. Growth was measured by means of a direct cell counting method and pigment synthesis was photometrically assessed. Addition of glycine resulted in an exponential increase in biomass, but not in pigment production. Tryptophan as the sole nitrogen source caused distinct brown staining of the medium, without increasing biomass. Simultaneous equimolar addition of both amino acids resulted in an initial increase in biomass as a sign of preferential metabolism of glycine, followed by a growth plateau and pigment production which, caused by higher biomass, occurred more rapidly than after addition of tryptophan alone. The yeast-cell morphology changed from round to oval. Addition of glycine to the tryptophan-containing liquid culture stopped pigment formation with simultaneous growth induction. These in vitro on-off phenomena depending on the nitrogen source might be significant in the pathogenesis of pityriasis versicolor: hyperhidrosis followed by preferential consumption of individual nitrogen sources such as glycine with exponential growth and thereafter transamination of tryptophan and TRP-dependent pigment synthesis.

  6. Regulation of protein synthesis during photomorphogenesis of gametophytes of the fern Onoclea sensibilis

    SciTech Connect

    Chansa-Ngavej, K.; Raghavan, V. )

    1989-08-01

    Gametophytes of the fern Onoclea sensibilis grow as filaments in the dark and in red light and become planar in blue light. Pulse-labeling 4-day-old gametophytes with ({sup 35}S)methionine at different times after transfer to dark, red, and blue light environments revealed higher rates of amino acid uptake and protein synthesis in blue light than in red light or in the dark. Characterization of the extant and newly synthesized soluble proteins by one- and two-dimensional gel electrophoresis showed that the patterns of protein accumulation and synthesis in gametophytes exposed to short periods of red or blue light were qualitatively indistinguishable from those of gametophytes maintained in the dark. However, some striking increases and decreases in the levels of certain polypeptides were noted and these changes were accentuated during continued growth of gametophytes in the different environments. The results show that photomorphogenesis of gametophytes of O. sensibilis is associated with quantitative rather than qualitative changes in the population of mRNAs available for translation.

  7. The global regulator LaeA controls penicillin biosynthesis, pigmentation and sporulation, but not roquefortine C synthesis in Penicillium chrysogenum.

    PubMed

    Kosalková, Katarina; García-Estrada, Carlos; Ullán, Ricardo V; Godio, Ramiro P; Feltrer, Raúl; Teijeira, Fernando; Mauriz, Elba; Martín, Juan Francisco

    2009-02-01

    The biosynthesis of the beta-lactam antibiotic penicillin is an excellent model for the study of secondary metabolites produced by filamentous fungi due to the good background knowledge on the biochemistry and molecular genetics of the beta-lactam producing microorganisms. The three genes (pcbAB, pcbC, penDE) encoding enzymes of the penicillin pathway in Penicillium chrysogenum are clustered, but no penicillin pathway-specific regulators have been found in the genome region that contains the penicillin gene cluster. The biosynthesis of this beta-lactam is controlled by global regulators of secondary metabolism rather than by a pathway-specific regulator. In this work we have identified the gene encoding the secondary metabolism global regulator LaeA in P. chrysogenum (PcLaeA), a nuclear protein with a methyltransferase domain. The PclaeA gene is present as a single copy in the genome of low and high-penicillin producing strains and is not located in the 56.8-kb amplified region occurring in high-penicillin producing strains. Overexpression of the PclaeA gene gave rise to a 25% increase in penicillin production. PclaeA knock-down mutants exhibited drastically reduced levels of penicillin gene expression and antibiotic production and showed pigmentation and sporulation defects, but the levels of roquefortine C produced and the expression of the dmaW involved in roquefortine biosynthesis remained similar to those observed in the wild-type parental strain. The lack of effect on the synthesis of roquefortine is probably related to the chromatin arrangement in the low expression roquefortine promoters as compared to the bidirectional pbcAB-pcbC promoter region involved in penicillin biosynthesis. These results evidence that PcLaeA not only controls some secondary metabolism gene clusters, but also asexual differentiation in P. chrysogenum.

  8. MiR-30a-3p Negatively Regulates BAFF Synthesis in Systemic Sclerosis and Rheumatoid Arthritis Fibroblasts

    PubMed Central

    Philippe, Lucas; Gong, Ya-Zhuo; Bahram, Seiamak; Cetin, Semih; Pfeffer, Sébastien; Gottenberg, Jacques-Eric; Wachsmann, Dominique; Georgel, Philippe; Sibilia, Jean

    2014-01-01

    We evaluated micro (mi) RNA-mediated regulation of BAFF expression in fibroblasts using two concomitant models: (i) synovial fibroblasts (FLS) isolated from healthy controls (N) or Rheumatoid Arthritis (RA) patients; (ii) human dermal fibroblasts (HDF) isolated from healthy controls (N) or Systemic Sclerosis (SSc) patients. Using RT-qPCR and ELISA, we first showed that SScHDF synthesized and released BAFF in response to Poly(I:C) or IFN-γ treatment, as previously observed in RAFLS, whereas NHDF released BAFF preferentially in response to IFN-γ. Next, we demonstrated that miR-30a-3p expression was down regulated in RAFLS and SScHDF stimulated with Poly(I:C) or IFN-γ. Moreover, we demonstrated that transfecting miR-30a-3p mimic in Poly(I:C)- and IFN-γ-activated RAFLS and SScHDF showed a strong decrease on BAFF synthesis and release and thus B cells survival in our model. Interestingly, FLS and HDF isolated from healthy subjects express higher levels of miR-30a-3p and lower levels of BAFF than RAFLS and SScHDF. Transfection of miR-30a-3p antisense in Poly(I:C)- and IFN-γ-activated NFLS and NHDF upregulated BAFF secretion, confirming that this microRNA is a basal repressors of BAFF expression in cells from healthy donors. Our data suggest a critical role of miR-30a-3p in the regulation of BAFF expression, which could have a major impact in the regulation of the autoimmune responses occurring in RA and SSc. PMID:25360821

  9. Phosphodiesterase A1, a regulator of cellulose synthesis in Acetobacter xylinum, is a heme-based sensor.

    PubMed

    Chang, A L; Tuckerman, J R; Gonzalez, G; Mayer, R; Weinhouse, H; Volman, G; Amikam, D; Benziman, M; Gilles-Gonzalez, M A

    2001-03-27

    The phosphodiesterase A1 protein of Acetobacter xylinum, AxPDEA1, is a key regulator of bacterial cellulose synthesis. This phosphodiesterase linearizes cyclic bis(3'-->5')diguanylic acid, an allosteric activator of the bacterial cellulose synthase, to the ineffectual pGpG. Here we show that AxPDEA1 contains heme and is regulated by reversible binding of O(2) to the heme. Apo-AxPDEA1 has less than 2% of the phosphodiesterase activity of holo-AxPDEA1, and reconstitution with hemin restores full activity. O(2) regulation is due to deoxyheme being a better activator than oxyheme. AxPDEA1 is homologous to the Escherichia coli direct oxygen sensor protein, EcDos, over its entire length and is homologous to the FixL histidine kinases over only a heme-binding PAS domain. The properties of the heme-binding domain of AxPDEA1 are significantly different from those of other O(2)-responsive heme-based sensors. The rate of AxPDEA1 autoxidation (half-life > 12 h) is the slowest observed so far for this type of heme protein fold. The O(2) affinity of AxPDEA1 (K(d) approximately 10 microM) is comparable to that of EcDos, but the rate constants for O(2) association (k(on) = 6.6 microM(-)(1) s(-)(1)) and dissociation (k(off) = 77 s(-)(1)) are 2000 times higher. Our results illustrate the versatility of signal transduction mechanisms for the heme-PAS class of O(2) sensors and provide the first example of O(2) regulation of a second messenger.

  10. Loss of Inositol Phosphorylceramide Sphingolipid Mannosylation Induces Plant Immune Responses and Reduces Cellulose Content in Arabidopsis[OPEN

    PubMed Central

    Ishikawa, Toshiki; Rennie, Emilie A.; Lao, Jeemeng; Yan, Jingwei; Tsai, Alex Yi-Lin; Baidoo, Edward E.K.; Demura, Taku; Kawai-Yamada, Maki

    2016-01-01

    Glycosylinositol phosphorylceramides (GIPCs) are a class of glycosylated sphingolipids found in plants, fungi, and protozoa. These lipids are abundant in the plant plasma membrane, forming ∼25% of total plasma membrane lipids. Little is known about the function of the glycosylated headgroup, but two recent studies have indicated that they play a key role in plant signaling and defense. Here, we show that a member of glycosyltransferase family 64, previously named ECTOPICALLY PARTING CELLS1, is likely a Golgi-localized GIPC-specific mannosyl-transferase, which we renamed GIPC MANNOSYL-TRANSFERASE1 (GMT1). Sphingolipid analysis revealed that the Arabidopsis thaliana gmt1 mutant almost completely lacks mannose-carrying GIPCs. Heterologous expression of GMT1 in Saccharomyces cerevisiae and tobacco (Nicotiana tabacum) cv Bright Yellow 2 resulted in the production of non-native mannosylated GIPCs. gmt1 displays a severe dwarfed phenotype and a constitutive hypersensitive response characterized by elevated salicylic acid and hydrogen peroxide levels, similar to that we previously reported for the Golgi-localized, GIPC-specific, GDP-Man transporter GONST1 (Mortimer et al., 2013). Unexpectedly, we show that gmt1 cell walls have a reduction in cellulose content, although other matrix polysaccharides are unchanged. PMID:27895225

  11. Fumonisin B1 (FB1) Induces Lamellar Separation and Alters Sphingolipid Metabolism of In Vitro Cultured Hoof Explants

    PubMed Central

    Reisinger, Nicole; Dohnal, Ilse; Nagl, Veronika; Schaumberger, Simone; Schatzmayr, Gerd; Mayer, Elisabeth

    2016-01-01

    One of the most important hoof diseases is laminitis. Yet, the pathology of laminitis is not fully understood. Different bacterial toxins, e.g. endotoxins or exotoxins, seem to play an important role. Additionally, ingestion of mycotoxins, toxic secondary metabolites of fungi, might contribute to the onset of laminitis. In this respect, fumonsins are of special interest since horses are regarded as species most susceptible to this group of mycotoxins. The aim of our study was to investigate the influence of fumonisin B1 (FB1) on primary isolated epidermal and dermal hoof cells, as well as on the lamellar tissue integrity and sphingolipid metabolism of hoof explants in vitro. There was no effect of FB1 at any concentration on dermal or epidermal cells. However, FB1 significantly reduced the separation force of explants after 24 h of incubation. The Sa/So ratio was significantly increased in supernatants of explants incubated with FB1 (2.5–10 µg/mL) after 24 h. Observed effects on Sa/So ratio were linked to significantly increased sphinganine concentrations. Our study showed that FB1 impairs the sphingolipid metabolism of explants and reduces lamellar integrity at non-cytotoxic concentrations. FB1 might, therefore, affect hoof health. Further in vitro and in vivo studies are necessary to elucidate the effects of FB1 on the equine hoof in more detail. PMID:27023602

  12. A combined fluorescence spectroscopy, confocal and 2-photon microscopy approach to re-evaluate the properties of sphingolipid domains.

    PubMed

    Pinto, Sandra N; Fernandes, Fábio; Fedorov, Alexander; Futerman, Anthony H; Silva, Liana C; Prieto, Manuel

    2013-09-01

    The aim of this study is to provide further insight about the interplay between important signaling lipids and to characterize the properties of the lipid domains formed by those lipids in membranes containing distinct composition. To this end, we have used a combination of fluorescence spectroscopy, confocal and two-photon microscopy and a stepwise approach to re-evaluate the biophysical properties of sphingolipid domains, particularly lipid rafts and ceramide (Cer)-platforms. By using this strategy we were able to show that, in binary mixtures, sphingolipids (Cer and sphingomyelin, SM) form more tightly packed gel domains than those formed by phospholipids with similar acyl chain length. In more complex lipid mixtures, the interaction between the different lipids is intricate and is strongly dictated by the Cer-to-Chol ratio. The results show that in quaternary phospholipid/SM/Chol/Cer mixtures, Cer forms gel domains that become less packed as Chol is increased. Moreover, the extent of gel phase formation is strongly reduced in these mixtures, even though Cer molar fraction is increased. These results suggest that in biological membranes, lipid domains such as rafts and ceramide platforms, might display distinctive biophysical properties depending on the local lipid composition at the site of the membrane where they are formed, further highlighting the potential role of membrane biophysical properties as an underlying mechanism for mediating specific biological processes.

  13. Altering the Mitochondrial Fatty Acid Synthesis (mtFASII) Pathway Modulates Cellular Metabolic States and Bioactive Lipid Profiles as Revealed by Metabolomic Profiling

    PubMed Central

    Clay, Hayley B.; Parl, Angelika K.; Mitchell, Sabrina L.; Singh, Larry; Bell, Lauren N.; Murdock, Deborah G.

    2016-01-01

    Despite the presence of a cytosolic fatty acid synthesis pathway, mitochondria have retained their own means of creating fatty acids via the mitochondrial fatty acid synthesis (mtFASII) pathway. The reason for its conservation has not yet been elucidated. Therefore, to better understand the role of mtFASII in the cell, we used thin layer chromatography to characterize the contribution of the mtFASII pathway to the fatty acid composition of selected mitochondrial lipids. Next, we performed metabolomic analysis on HeLa cells in which the mtFASII pathway was either hypofunctional (through knockdown of mitochondrial acyl carrier protein, ACP) or hyperfunctional (through overexpression of mitochondrial enoyl-CoA reductase, MECR). Our results indicate that the mtFASII pathway contributes little to the fatty acid composition of mitochondrial lipid species examined. Additionally, loss of mtFASII function results in changes in biochemical pathways suggesting alterations in glucose utilization and redox state. Interestingly, levels of bioactive lipids, including lysophospholipids and sphingolipids, directly correlate with mtFASII function, indicating that mtFASII may be involved in the regulation of bioactive lipid levels. Regulation of bioactive lipid levels by mtFASII implicates the pathway as a mediator of intracellular signaling. PMID:26963735

  14. Enhanced killing of SCC17B human head and neck squamous cell carcinoma cells after photodynamic therapy plus fenretinide via the de novo sphingolipid biosynthesis pathway and apoptosis.

    PubMed

    Boppana, Nithin B; Stochaj, Ursula; Kodiha, Mohamed; Bielawska, Alicja; Bielawski, Jacek; Pierce, Jason S; Korbelik, Mladen; Separovic, Duska

    2015-05-01

    Because photodynamic therapy (PDT) alone is not always effective as an anticancer treatment, PDT is combined with other anticancer agents for improved efficacy. The clinically-relevant fenretinide [N-(4-hydroxyphenyl) retinamide; 4HPR], was combined with the silicon phthalocyanine photosensitizer Pc4-mediated PDT to test for their potential to enhance killing of SCC17B cells, a clinically-relevant model of human head and neck squamous cell carcinoma. Because each of these treatments induces apoptosis and regulates the de novo sphingolipid (SL) biosynthesis pathway, the role of ceramide synthase, the pathway-associated enzyme, in PDT+4HPR-induced apoptotic cell death was determined using the ceramide synthase inhibitor fumonisin B1 (FB). PDT+4HPR enhanced loss of clonogenicity. zVAD-fmk, a pan-caspase inhibitor, and FB, protected cells from death post-PDT+4HPR. In contrast, the anti-apoptotic protein Bcl2 inhibitor ABT199 enhanced cell killing after PDT+4HPR. Combining PDT with 4HPR led to FB-sensitive, enhanced Bax associated with mitochondria and cytochrome c redistribution. Mass spectrometry data showed that the accumulation of C16-dihydroceramide, a precursor of ceramide in the de novo SL biosynthesis pathway, was enhanced after PDT+4HPR. Using quantitative confocal microscopy, we found that PDT+4HPR enhanced dihydroceramide/ceramide accumulation in the ER, which was inhibited by FB. The results suggest that SCC17B cells are sensitized to PDT by 4HPR via the de novo SL biosynthesis pathway and apoptosis, and imply potential clinical relevance of the combination for cancer treatment.

  15. Enhanced killing of SCC17B human head and neck squamous cell carcinoma cells after photodynamic therapy plus fenretinide via the de novo sphingolipid biosynthesis pathway and apoptosis

    PubMed Central

    BOPPANA, NITHIN B.; STOCHAJ, URSULA; KODIHA, MOHAMED; BIELAWSKA, ALICJA; BIELAWSKI, JACEK; PIERCE, JASON S.; KORBELIK, MLADEN; SEPAROVIC, DUSKA

    2015-01-01

    Because photodynamic therapy (PDT) alone is not always effective as an anticancer treatment, PDT is combined with other anticancer agents for improved efficacy. The clinically-relevant fenretinide [N-(4-hydroxyphenyl) retinamide; 4HPR], was combined with the silicon phthalocyanine photosensitizer Pc4-mediated PDT to test for their potential to enhance killing of SCC17B cells, a clinically-relevant model of human head and neck squamous cell carcinoma. Because each of these treatments induces apoptosis and regulates the de novo sphingolipid (SL) biosynthesis pathway, the role of ceramide synthase, the pathway-associated enzyme, in PDT+4HPR-induced apoptotic cell death was determined using the ceramide synthase inhibitor fumonisin B1 (FB). PDT+4HPR enhanced loss of clonogenicity. zVAD-fmk, a pan-caspase inhibitor, and FB, protected cells from death post-PDT+4HPR. In contrast, the anti-apoptotic protein Bcl2 inhibitor ABT199 enhanced cell killing after PDT+4HPR. Combining PDT with 4HPR led to FB-sensitive, enhanced Bax associated with mitochondria and cytochrome c redistribution. Mass spectrometry data showed that the accumulation of C16-dihydroceramide, a precursor of ceramide in the de novo SL biosynthesis pathway, was enhanced after PDT+4HPR. Using quantitative confocal microscopy, we found that PDT+4HPR enhanced dihydroceramide/ceramide accumulation in the ER, which was inhibited by FB. The results suggest that SCC17B cells are sensitized to PDT by 4HPR via the de novo SL biosynthesis pathway and apoptosis, and imply potential clinical relevance of the combination for cancer treatment. PMID:25739041

  16. Synthesis of a new conjugated polymer for DNA alkylation and gene regulation.

    PubMed

    Nie, Chenyao; Zhu, Chunlei; Feng, Liheng; Lv, Fengting; Liu, Libing; Wang, Shu

    2013-06-12

    A new polyfluorene derivative containing pendent alkylating chlorambucil (PFP-Cbl) was synthesized and characterized. Under direct incubation with DNA in vitro, PFP-Cbl could undergo an efficient DNA alkylating reaction and induce DNA cross-linking. In vitro transcription and translation experiment exhibited that the PFP-Cbl significantly down-regulated the gene expression of luciferase reporter plasmid. The down-regulation of gene expression was also verified through the transfection experiment of p-EGFP plasmid, which showed decreased green fluorescent protein (GFP) in cells. Meanwhile, the self-luminous property of PFP-Cbl could make it able to trace the internalized PFP-Cbl and plasmid complexes resulted from cross-linking in cells by fluorescent microscopy. Combining the features of alkylating function, multivalent binding sites, and fluorescent characteristics, PFP-Cbl provides a new insight in the area of gene regulation and extends the new applications of conjugated polymers (CPs).

  17. Lipin-1 regulation of phospholipid synthesis maintains endoplasmic reticulum homeostasis and is critical for triple-negative breast cancer cell survival.

    PubMed

    He, Jingquan; Zhang, Feng; Tay, Li Wei Rachel; Boroda, Salome; Nian, Weiqi; Levental, Kandice R; Levental, Ilya; Harris, Thurl E; Chang, Jeffrey T; Du, Guangwei

    2017-03-27

    Cancer cells reprogram their metabolism to increase the synthesis of macromolecules for rapid proliferation. Compared to fatty acids, much less is known about the synthesis of phospholipids, which is essential for membrane biogenesis in cancer cells. We found that LPIN1, which encodes lipin-1, a phosphatidic acid phosphatase (PAP) controlling the rate-limiting step in the phospholipid synthesis pathway, is highly up-regulated in basal-like triple-negative breast cancer (TNBC). Moreover, high LPIN1 expression correlates with the poor prognosis of these patients. Knockdown of LPIN1 increases apoptosis in basal-like TNBC cell lines, whereas it has minimal or less effect on normal human mammary gland epithelial cells (HMECs) and estrogen receptor-positive breast cancer cell lines. Fatty acid incorporation and lipidomics analyses showed that LPIN1 knockdown blocks phospholipid synthesis and changes membrane lipid compositions that ultimately induce the activation of 1 of the 3 branches of unfolded protein responses, the inositol-requiring enzyme-1α pathway. We also show for the first time, to our knowledge, that lipin-1 knockdown significantly inhibits tumor growth in vivo using an orthotopic xenograft breast mouse model. Our results suggest that lipin-1 is a potential target for cancer therapy.-He, J., Zhang, F., Tay, L. W. R., Boroda, S., Nian, W., Levental, K. R., Levental, I., Harris, T. E., Chang, J. T., Du, G. Lipin-1 regulation of phospholipid synthesis maintains endoplasmic reticulum homeostasis and is critical for triple-negative breast cancer cell survival.

  18. Regulation of chloroplast number and DNA synthesis in higher plants. Final report

    SciTech Connect

    Mullet, J.E.

    1995-11-10

    The long term objective of this research is to understand the process of chloroplast development and its coordination with leaf development in higher plants. This is important because the photosynthetic capacity of plants is directly related to leaf and chloroplast development. This research focuses on obtaining a detailed description of leaf development and the early steps in chloroplast development including activation of plastid DNA synthesis, changes in plastid DNA copy number, activation of chloroplast transcription and increases in plastid number per cell. The grant will also begin analysis of specific biochemical mechanisms by isolation of the plastid DNA polymerase, and identification of genetic mutants which are altered in their accumulation of plastid DNA and plastid number per cell.

  19. Regulation of chloroplast number and DNA synthesis in higher plants. Final report

    SciTech Connect

    Mullet, J.E.

    1995-11-10

    The long term objective of this research is to understand the process of chloroplast development and its coordination with leaf development in higher plants. This is important because the photosynthetic capacity of plants is directly related to leaf and chloroplast development. This research focuses on obtaining a detailing description of leaf development and the early steps in chloroplast development including activation of plastid DNA synthesis, changes in plastid DNA copy number, activation of chloroplast transcription and increases in plastid number per cell. The grant will also begin analysis of specific biochemical mechanisms by isolation of the plastid DNA polymerase, and identification of genetic mutants which are altered in their accumulation of plastid DNA and plastid number per cell.

  20. Divergence in enzyme regulation between Caenorhabditis elegans and human tyrosine hydroxylase, the key enzyme in the synthesis of dopamine.

    PubMed

    Calvo, Ana C; Pey, Angel L; Miranda-Vizuete, Antonio; Døskeland, Anne P; Martinez, Aurora

    2011-02-15

    TH (tyrosine hydroxylase) is the rate-limiting enzyme in the synthesis of catecholamines. The cat-2 gene of the nematode Caenorhabditis elegans is expressed in mechanosensory dopaminergic neurons and has been proposed to encode a putative TH. In the present paper, we report the cloning of C. elegans full-length cat-2 cDNA and a detailed biochemical characterization of the encoded CAT-2 protein. Similar to other THs, C. elegans CAT-2 is composed of an N-terminal regulatory domain followed by a catalytic domain and a C-terminal oligomerization domain and shows high substrate specificity for L-tyrosine. Like hTH (human TH), CAT-2 is tetrameric and is phosphorylated at Ser35 (equivalent to Ser40 in hTH) by PKA (cAMP-dependent protein kinase). However, CAT-2 is devoid of characteristic regulatory mechanisms present in hTH, such as negative co-operativity for the cofactor, substrate inhibition or feedback inhibition exerted by catecholamines, end-products of the pathway. Thus TH activity in C. elegans displays a weaker regulation in comparison with the human orthologue, resembling a constitutively active enzyme. Overall, our data suggest that the intricate regulation characteristic of mammalian TH might have evolved from more simple models to adjust to the increasing complexity of the higher eukaryotes neuroendocrine systems.

  1. The laforin-malin complex negatively regulates glycogen synthesis by modulating cellular glucose uptake via glucose transporters.

    PubMed

    Singh, Pankaj Kumar; Singh, Sweta; Ganesh, Subramaniam

    2012-02-01

    Lafora disease (LD), an inherited and fatal neurodegenerative disorder, is characterized by increased cellular glycogen content and the formation of abnormally branched glycogen inclusions, called Lafora bodies, in the affected tissues, including neurons. Therefore, laforin phosphatase and malin ubiquitin E3 ligase, the two proteins that are defective in LD, are thought to regulate glycogen synthesis through an unknown mechanism, the defects in which are likely to underlie some of the symptoms of LD. We show here that laforin's subcellular localization is dependent on the cellular glycogen content and that the stability of laforin is determined by the cellular ATP level, the activity of 5'-AMP-activated protein kinase, and the affinity of malin toward laforin. By using cell and animal models, we further show that the laforin-malin complex regulates cellular glucose uptake by modulating the subcellular localization of glucose transporters; loss of malin or laforin resulted in an increased abundance of glucose transporters in the plasma membrane and therefore excessive glucose uptake. Loss of laforin or malin, however, did not affect glycogen catabolism. Thus, the excessive cellular glucose level appears to be the primary trigger for the abnormally higher levels of cellular glycogen seen in LD.

  2. Synthesis and evaluation of tamoxifen derivatives with a long alkyl side chain as selective estrogen receptor down-regulators.

    PubMed

    Shoda, Takuji; Kato, Masashi; Harada, Rintaro; Fujisato, Takuma; Okuhira, Keiichiro; Demizu, Yosuke; Inoue, Hideshi; Naito, Mikihiko; Kurihara, Masaaki

    2015-07-01

    Estrogen receptors (ERs) play a major role in the growth of human breast cancer cells. An antagonist that acts as not only an inhibitor of ligand binding but also an inducer of the down-regulation of ER would be useful for the treatment for ER-positive breast cancer. We previously reported the design and synthesis of a selective estrogen receptor down-regulator (SERD), (E/Z)-4-(1-{4-[2-(dodecylamino)ethoxy]phenyl}-2-phenylbut-1-en-1-yl)phenol (C12), which is a tamoxifen derivative having a long alkyl chain on the amine moiety. This compound induced degradation of ERα via a proteasome-dependent pathway and showed an antagonistic effect in MCF-7 cells. With the aim of increasing the potency of SERDs, we designed and synthesized various tamoxifen derivatives that have various lengths and terminal groups of the long alkyl side chain. During the course of our investigation, C10F having a 10-fluorodecyl group on the amine moiety of 4-OHT was shown to be the most potent compound among the tamoxifen derivatives. Moreover, computational docking analysis suggested that the long alkyl chain interacted with the hydrophobic region on the surface of the ER, which is a binding site of helix 12 and coactivator. These results provide useful information to develop promising candidates as SERDs.

  3. Identification of the Main Regulator Responsible for Synthesis of the Typical Yellow Pigment Produced by Trichoderma reesei.

    PubMed

    Derntl, Christian; Rassinger, Alice; Srebotnik, Ewald; Mach, Robert L; Mach-Aigner, Astrid R

    2016-10-15

    The industrially used ascomycete Trichoderma reesei secretes a typical yellow pigment during cultivation, while other Trichoderma species do not. A comparative genomic analysis suggested that a putative secondary metabolism cluster, containing two polyketide-synthase encoding genes, is responsible for the yellow pigment synthesis. This cluster is conserved in a set of rather distantly related fungi, including Acremonium chrysogenum and Penicillium chrysogenum In an attempt to silence the cluster in T. reesei, two genes of the cluster encoding transcription factors were individually deleted. For a complete genetic proof-of-function, the genes were reinserted into the genomes of the respective deletion strains. The deletion of the first transcription factor (termed yellow pigment regulator 1 [Ypr1]) resulted in the full abolishment of the yellow pigment formation and the expression of most genes of this cluster. A comparative high-pressure liquid chromatography (HPLC) analysis of supernatants of the ypr1 deletion and its parent strain suggested the presence of several yellow compounds in T. reesei that are all derived from the same cluster. A subsequent gas chromatography/mass spectrometry analysis strongly indicated the presence of sorbicillin in the major HPLC peak. The presence of the second transcription factor, termed yellow pigment regulator 2 (Ypr2), reduces the yellow pigment formation and the expression of most cluster genes, including the gene encoding the activator Ypr1.

  4. Ca²⁺ regulation of mitochondrial ATP synthesis visualized at the single cell level.

    PubMed

    Nakano, Masahiro; Imamura, Hiromi; Nagai, Takeharu; Noji, Hiroyuki

    2011-07-15

    Intracellular Ca(2+) levels play a crucial role in the control of ATP synthesis. However, the spatiotemporal correlation between ATP and Ca(2+) remains unclear due to the inability to visualize these factors within same individual cells. A Förster resonance energy transfer (FRET)-based fluorescent ATP probe, named ATeam, was recently developed for ATP imaging in single living cells. However, the spectra of cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) used as the FRET donor and the acceptor, respectively, significantly overlap with the ultraviolet-excitable Ca(2+) probe, fura-2. In the present work, we developed new red-shifted ATP probes, GO-ATeams, in which green fluorescent protein (GFP) and orange fluorescent protein (OFP) was used as the FRET pair to minimize spectral overlap with the fura-2 emission. The dynamics of intracellular Ca(2+) and mitochondrial ATP levels in single histamine-stimulated HeLa cells were successfully visualized by using fura-2 and GO-ATeam. The experiments showed that histamine induced increases of both intracellular Ca(2+) and mitochondrial ATP levels. The increment of mitochondrial ATP levels was proportional to that of Ca(2+). This finding suggests that cellular Ca(2+) levels might precisely control mitochondrial ATP synthesis in response to the increased ATP consumption triggered by Ca(2+). In addition, GO-ATeam has several advantages over the original ATeam. The GO-ATeam signal was more stable against acidification, which would allow ATP imaging inside acidic intracellular compartments. Also, the GO-ATeam excitation wavelength is much less phototoxic to cells, making the probe suitable for long-time observation.

  5. Integrin-dependent Control of Translation: Engagement of Integrin αIIbβ3 Regulates Synthesis of Proteins in Activated Human Platelets

    PubMed Central

    Pabla, Ravinder; Weyrich, Andrew S.; Dixon, Dan A.; Bray, Paul F.; McIntyre, Thomas M.; Prescott, Stephen M.; Zimmerman, Guy A.

    1999-01-01

    Integrins are widely expressed plasma membrane adhesion molecules that tether cells to matrix proteins and to one another in cell–cell interactions. Integrins also transmit outside-in signals that regulate functional responses of cells, and are known to influence gene expression by regulating transcription. In previous studies we found that platelets, which are naturally occurring anucleate cytoplasts, translate preformed mRNA transcripts when they are activated by outside-in signals. Using strategies that interrupt engagement of integrin αIIbβ3 by fibrinogen and platelets deficient in this integrin, we found that αIIbβ3 regulates the synthesis of B cell lymphoma 3 (Bcl-3) when platelet aggregation is induced by thrombin. We also found that synthesis of Bcl-3, which occurs via a specialized translation control pathway regulated by mammalian target of rapamycin (mTOR), is induced when platelets adhere to immobilized fibrinogen in the absence of thrombin and when integrin αIIbβ3 is engaged by a conformation-altering antibody against integrin αIIbβ3. Thus, outside-in signals delivered by integrin αIIbβ3 are required for translation of Bcl-3 in thrombin-stimulated aggregated platelets and are sufficient to induce translation of this marker protein in the absence of thrombin. Engagement of integrin α2β1 by collagen also triggered synthesis of Bcl-3. Thus, control of translation may be a general mechanism by which surface adhesion molecules regulate gene expression. PMID:9885253

  6. The pvc Gene Cluster of Pseudomonas aeruginosa: Role in Synthesis of the Pyoverdine Chromophore and Regulation by PtxR and PvdS

    PubMed Central

    Stintzi, Alain; Johnson, Zaiga; Stonehouse, Martin; Ochsner, Urs; Meyer, Jean-Marie; Vasil, Michael L.; Poole, Keith

    1999-01-01

    A putative operon of four genes implicated in the synthesis of the chromophore moiety of the Pseudomonas aeruginosa siderophore pyoverdine, dubbed pvcABCD (where pvc stands for pyoverdine chromophore), was cloned and sequenced. Mutational inactivation of the pvc genes abrogated pyoverdine biosynthesis, consistent with their involvement in the biosynthesis of this siderophore. pvcABCD expression was negatively regulated by iron and positively regulated by both PvdS, the alternate sigma factor required for pyoverdine biosynthesis, and PtxR, a LysR family activator previously implicated in exotoxin A regulation. PMID:10383985

  7. Synthesis and regulation of chlorogenic acid in potato: Rerouting phenylpropanoid flux in HQT silenced lines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chlorogenic acid (CGA) is the major phenolic sink in potato tubers and can constitute over 90% of total phenylpropanoids. The regulation of CGA biosynthesis in potato and the role of the CGA biosynthetic gene hydroxycinnamoyl-CoA:quinate hydroxycinnamoyl transferase (HQT) was characterized. A sucros...

  8. Regulation of polyamine synthesis in plants. Final progress report, July 1, 1991--December 31, 1994

    SciTech Connect

    Malmberg, R.L.

    1995-07-01

    This research focused on unusual post-translational modifications occuring in a arginine decarboxylase cDNA clone in oats. A novel regulatory mechanism for polyamines was explored and an attempt was made to characterize it. A plant ornithine decarboxylase cDNA was identified in Arabidopsis. Further work remains on the mechanisms of polyamine regulation and function in plants.

  9. Sphingolipids Containing Very-Long-Chain Fatty Acids Define a Secretory Pathway for Specific Polar Plasma Membrane Protein Targeting in Arabidopsis[W

    PubMed Central

    Markham, Jonathan E.; Molino, Diana; Gissot, Lionel; Bellec, Yannick; Hématy, Kian; Marion, Jessica; Belcram, Katia; Palauqui, Jean-Christophe; Satiat-JeuneMaître, Béatrice; Faure, Jean-Denis

    2011-01-01

    Sphingolipids are a class of structural membrane lipids involved in membrane trafficking and cell polarity. Functional analysis of the ceramide synthase family in Arabidopsis thaliana demonstrates the existence of two activities selective for the length of the acyl chains. Very-long-acyl-chain (C > 18 carbons) but not long-chain sphingolipids are essential for plant development. Reduction of very-long-chain fatty acid sphingolipid levels leads in particular to auxin-dependent inhibition of lateral root emergence that is associated with selective aggregation of the plasma membrane auxin carriers AUX1 and PIN1 in the cytosol. Defective targeting of polar auxin carriers is characterized by specific aggregation of Rab-A2a– and Rab-A1e–labeled early endosomes along the secretory pathway. These aggregates correlate with the accumulation of membrane structures and vesicle fragmentation in the cytosol. In conclusion, sphingolipids with very long acyl chains define a trafficking pathway with specific endomembrane compartments and polar auxin transport protein cargoes. PMID:21666002

  10. Age-dependent changes in the sphingolipid composition of CD4+ T cell membranes and immune synapses implicate glucosylceramides in age-related T cell dysfunction

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sphingolipid (SL4) composition can influence the biophysical properties of cell membranes. Additionally, specific SL modulate signaling pathways involved in proliferation, senescence, and apoptosis. We investigated age-dependent changes in the SL composition of CD4+ T cells, and the impact of these ...

  11. Na/K-ATPase signaling regulates collagen synthesis through microRNA-29b-3p in cardiac fibroblasts.

    PubMed

    Drummond, Christopher A; Hill, Michael C; Shi, Huilin; Fan, Xiaoming; Xie, Jeffrey X; Haller, Steven T; Kennedy, David J; Liu, Jiang; Garrett, Michael R; Xie, Zijian; Cooper, Christopher J; Shapiro, Joseph I; Tian, Jiang

    2016-03-01

    Chronic kidney disease (CKD) is accompanied by cardiac fibrosis, hypertrophy, and dysfunction, which are commonly referred to as uremic cardiomyopathy. Our previous studies found that Na/K-ATPase ligands or 5/6th partial nephrectomy (PNx) induces cardiac fibrosis in rats and mice. The current study used in vitro and in vivo models to explore novel roles for microRNA in this mechanism of cardiac fibrosis formation. To accomplish this, we performed microRNA profiling with RT-qPCR based arrays on cardiac tissue from rats subjected to marinobufagenin (MBG) infusion or PNx. The analysis showed that a series of fibrosis-related microRNAs were dysregulated. Among the dysregulated microRNAs, microRNA (miR)-29b-3p, which directly targets mRNA of collagen, was consistently reduced in both PNx and MBG-infused animals. In vitro experiments demonstrated that treatment of primary cultures of adult rat cardiac fibroblasts with Na/K-ATPase ligands induced significant increases in the fibrosis marker, collagen protein, and mRNA expression compared with controls, whereas miR-29b-3p expression decreased >50%. Transfection of miR-29b-3p mimics into cardiac fibroblasts inhibited cardiotonic steroids-induced collagen synthesis. Moreover, a specific Na/K-ATPase signaling antagonist, pNaKtide, prevented ouabain-induced increases in collagen synthesis and decreases in miR-29b-3p expression in these cells. In conclusion, these data are the first to indicate that signaling through Na/K-ATPase regulates miRNAs and specifically, miR-29b-3p expression both in vivo and in vitro. Additionally, these data indicate that miR-29b-3p expression plays an important role in the formation of cardiac fibrosis in CKD.

  12. Interleukin 10 regulates inflammatory cytokine synthesis to protect against lipopolysaccharide-induced abortion and fetal growth restriction in mice.

    PubMed

    Robertson, Sarah A; Care, Alison S; Skinner, Rebecca J

    2007-05-01

    Interleukin 10 (IL10) is a potent immune-regulating cytokine and inhibitor of inflammatory cytokine synthesis. To evaluate the anti-inflammatory role of IL10 in pregnancy, the response of genetically IL10-deficient mice to low-dose lipopolysaccharide (LPS)-induced abortion was examined. When IL10-null mutant C57Bl/6 (Il10(-/-)) and control (Il10(+/+)) mice were administered low-dose LPS on Day 9.5 of gestation, IL10 deficiency predisposed to fetal loss accompanied by growth restriction in remaining viable fetuses, with an approximately 10-fold reduction in the threshold dose for 100% abortion. After LPS administration, inflammatory cytokines tumor necrosis factor-alpha (TNFA) and IL6 were markedly increased in serum, uterine, and conceptus tissues in Il10(-/-) mice compared with Il10(+/+) mice, with elevated local synthesis of Tnfa and Il6 mRNAs in the gestational tissues. IL1A and IL12p40 were similarly elevated in serum and gestational tissues, whereas interferon gamma (IFNG) and soluble TNFRII content were unchanged in the absence of IL10. Recombinant IL10 rescued the increased susceptibility to LPS-induced fetal loss in Il10(-/-) mice but did not improve outcomes in Il10(+/+) mice. IL10 genotype also influenced the responsiveness of mice to a TNFA antagonist, etanercept. Fetal loss in Il10(-/-) mice was partly alleviated by moderate or high doses of etanercept, whereas Il10(+/+) mice were refractory to high-dose etanercept, consistent with attenuation by IL10 status of TNFA bioavailability after etanercept treatment. These data show that IL10 modulates resistance to inflammatory stimuli by downregulating expression of proinflammatory cytokines TNFA, IL6, IL1A, and IL12, acting to protect against inflammation-induced pathology in the implantation site.

  13. Regulation of leukotriene and 5oxoETE synthesis and the effect of 5-lipoxygenase inhibitors: a mathematical modeling approach

    PubMed Central

    2012-01-01

    Background 5-lipoxygenase (5-LO) is a key enzyme in the synthesis of leukotrienes and 5-Oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid (oxoETE). These inflammatory signaling molecules play a role in the pathology of asthma and so 5-LO inhibition is a promising target for asthma therapy. The 5-LO redox inhibitor zileuton (Zyflo IR/CR®) is currently marketed for the treatment of asthma in adults and children, but widespread use of zileuton is limited by its efficacy/safety profile, potentially related to its redox characteristics. Thus, a quantitative, mechanistic description of its functioning may be useful for development of improved anti-inflammatory targeting this mechanism. Results A mathematical model describing the operation of 5-LO, phospholipase A2, glutathione peroxidase and 5-hydroxyeicosanoid dehydrogenase was developed. The catalytic cycles of the enzymes were reconstructed and kinetic parameters estimated on the basis of available experimental data. The final model describes each stage of cys-leukotriene biosynthesis and the reactions involved in oxoETE production. Regulation of these processes by substrates (phospholipid concentration) and intracellular redox state (concentrations of reduced glutathione, glutathione (GSH), and lipid peroxide) were taken into account. The model enabled us to reveal differences between redox and non-redox 5-LO inhibitors under conditions of oxidative stress. Despite both redox and non-redox inhibitors suppressing leukotriene A4 (LTA4) synthesis, redox inhibitors are predicted to increase oxoETE production, thus compromising efficacy. This phenomena can be explained in terms of the pseudo-peroxidase activity of 5-LO and the ability of lipid peroxides to transform 5-LO into its active form even in the presence of redox inhibitors. Conclusions The mathematical model developed described quantitatively different mechanisms of 5-LO inhibition and simulations revealed differences between the potential therapeutic outcomes for these

  14. Regulation of polyamine synthesis in plants. Annual progress report, July 1, 1992--June 30, 1993

    SciTech Connect

    Malmberg, R.L.

    1995-07-01

    After isolation of a cDNA clone for the plant ARGdc, this research focused on unusual post-translational modifications occuring in a arginine decarboxylase cDNA clone in oats. A novel regulatory mechanism for polyamines was explored and an attempt was made to characterize it. A plant ornithine decarboxylase cDNA was identified in Arabidopsis. Further work remains on the mechanisms of polyamine regulation and function in plants.

  15. Differential regulation by cyclic AMP of starvation protein synthesis in Escherichia coli.

    PubMed

    Schultz, J E; Latter, G I; Matin, A

    1988-09-01

    Of the 30 carbon starvation proteins whose induction has been previously shown to be important for starvation survival of Escherichia coli, two-thirds were not induced in cya or crp deletion mutants of E. coli at the onset of carbon starvation. The rest were induced, although not necessarily with the same temporal pattern as exhibited in the wild type. The starvation proteins that were homologous to previously identified heat shock proteins belonged to the latter class and were hyperinduced in delta cya or delta crp mutants during starvation. Most of the cyclic AMP-dependent proteins were synthesized in the delta cya mutant if exogenous cyclic AMP was added at the onset of starvation. Furthermore, beta-galactosidase induction of several carbon starvation response gene fusions occurred only in a cya+ genetic background. Thus, two-thirds of the carbon starvation proteins of E. coli require cyclic AMP and its receptor protein for induction; the rest do not. The former class evidently has no role in starvation survival, since delta cya or delta crp mutants of either E. coli or Salmonella typhimurium survived starvation as well as their wild-type parents did. The latter class, therefore, is likely to have a direct role in starvation survival. This possibility is strengthened by the finding that nearly all of the cya- and crp-independent proteins were also induced during nitrogen starvation and, as shown previously, during phosphate starvation. Proteins whose synthesis is independent of cya- and crp control are referred to as Pex (postexponential).

  16. Protein synthesis and degradation are essential to regulate germline stem cell homeostasis in Drosophila testes.

    PubMed

    Yu, Jun; Lan, Xiang; Chen, Xia; Yu, Chao; Xu, Yiwen; Liu, Yujuan; Xu, Lingna; Fan, Heng-Yu; Tong, Chao

    2016-08-15

    The homeostasis of self-renewal and differentiation in stem cells is controlled by intrinsic signals and their niche. We conducted a large-scale RNA interference (RNAi) screen in Drosophila testes and identified 221 genes required for germline stem cell (GSC) maintenance or differentiation. Knockdown of these genes in transit-amplifying spermatogonia and cyst cells further revealed various phenotypes. Complex analysis uncovered that many of the identified genes are involved in key steps of protein synthesis and degradation. A group of genes that are required for mRNA splicing and protein translation contributes to both GSC self-renewal and early germ cell differentiation. Loss of genes in the protein degradation pathway in cyst cells leads to testis tumors consisting of overproliferated germ cells. Importantly, in the Cullin 4-RING E3 ubiquitin ligase (CRL4) complex, we identified multiple proteins that are crucial to GSC self-renewal: pic/DDB1, a CRL4 linker protein, is not only required for GSC self-renewal in flies but also for maintenance of spermatogonial stem cells (SSCs) in mice.

  17. Tyrosine phosphatases as key regulators of StAR induction and cholesterol transport: SHP2 as a potential tyrosine phosphatase involved in steroid synthesis.

    PubMed

    Cooke, Mariana; Mele, Pablo; Maloberti, Paula; Duarte, Alejandra; Poderoso, Cecilia; Orlando, Ulises; Paz, Cristina; Cornejo Maciel, Fabiana; Podestá, Ernesto J

    2011-04-10

    The phospho-dephosphorylation of intermediate proteins is a key event in the regulation of steroid biosynthesis. In this regard, it is well accepted that steroidogenic hormones act through the activation of serine/threonine (Ser/Thr) protein kinases. Although many cellular processes can be regulated by a crosstalk between different kinases and phosphatases, the relationship of Ser/Thr phosphorylation and tyrosine (Tyr)-dephosphorylation is a recently explored field in the regulation of steroid synthesis. Indeed in steroidogenic cells, one of the targets of hormone-induced Ser/Thr phosphorylation is a protein tyrosine phosphatase. Whereas protein tyrosine phosphatases were initially regarded as household enzymes with constitutive activity, dephosphorylating all the substrates they encountered, evidence is now accumulating that protein tyrosine phosphatases are tightly regulated by various mechanisms. Here, we will describe the role of protein tyrosine phosphatases in the regulation of steroid biosynthesis, relating them to steroidogenic acute regulatory protein, arachidonic acid metabolism and mitochondrial rearrangement.

  18. Regulation of hordein synthesis in barley high lysine mutant Notch-2.

    PubMed

    Tyagi, A; Santha, I M; Mehta, S L

    1992-02-01

    Genomic DNA isolated from barley cv. NP 113 and its high lysine mutant Notch-2, and restricted with different restriction enzymes was hybridized with B1 and C-hordein DNA probes. Similar Southern hybridization patterns were observed between NP 113 and Notch-2. Dot blot hybridization analysis of RNA isolated at different developmental stages and from different tissues of seed showed temporal as well as tissue specific expression. The results obtained indicate that regulation at the level of transcription/post transcription may be responsible for lower accumulation of hordein in mutant Notch-2.

  19. Proline metabolism in N2-fixing root nodules: energy transfer and regulation of purine synthesis.

    PubMed Central

    Kohl, D H; Schubert, K R; Carter, M B; Hagedorn, C H; Shearer, G

    1988-01-01

    N2-fixing root nodules of soybean (Glycine max L. Merr.) convert atmospheric N2 to ammonia(um) in an energy-intensive enzymatic reaction. These nodules synthesize large quantities of purines because nitrogen fixed by bacteria contained within this tissue is transferred to the shoots in the form of ureides, which are degradation products of purines. In animal systems, it has been proposed that proline biosynthesis by pyrroline-5-carboxylate reductase (P5CR) is used to generate the NADP+ required for the synthesis of the purine precursor ribose 5-phosphate. We have examined the levels, properties, and location of P5CR and proline dehydrogenase (ProDH) in soybean nodules. Nodule P5CR was found in the plant cytosol. Its activity was substantially higher than that reported for other animal and plant tissues and is 4-fold higher than in pea (Pisum sativum) nodules (which export amides). The Km for NADPH was lower by a factor of 25 than the Km for NADH, while the Vmax with NADPH was one-third of that with NADH. P5CR activity was diminished by NADP+ but not by proline. These characteristics are consistent with a role for P5CR in supporting nodule purine biosynthesis rather than in producing proline for incorporation into protein. ProDH activity was divided between the bacteroids and plant cytosol, but less than 2% was in the mitochondria-rich fractions. The specific activity of ProDH in soybean nodule bacteroids was comparable to that in rat liver mitochondria. In addition, we propose that some of the proline synthesized in the plant cytosol by P5CR is catabolized within the bacteroids by ProDH and that this represents a novel mechanism for transferring energy from the plant to its endosymbiont. PMID:3353366

  20. Akt Regulates TNFα Synthesis Downstream of RIP1 Kinase Activation during Necroptosis

    PubMed Central

    McNamara, Colleen R.; Ahuja, Ruchita; Osafo-Addo, Awo D.; Barrows, Douglas; Kettenbach, Arminja; Skidan, Igor; Teng, Xin; Cuny, Gregory D.; Gerber, Scott; Degterev, Alexei

    2013-01-01

    Necroptosis is a regulated form of necrotic cell death that has been implicated in the pathogenesis of various diseases including intestinal inflammation and systemic inflammatory response syndrome (SIRS). In this work, we investigated the signaling mechanisms controlled by the necroptosis mediator receptor interacting protein-1 (RIP1) kinase. We show that Akt kinase activity is critical for necroptosis in L929 cells and plays a key role in TNFα production. During necroptosis, Akt is activated in a RIP1 dependent fashion through its phosphorylation on Thr308. In L929 cells, this activation requires independent signaling inputs from both growth factors and RIP1. Akt controls necroptosis through downstream targeting of mammalian Target of Rapamycin complex 1 (mTORC1). Akt activity, mediated in part through mTORC1, links RIP1 to JNK activation and autocrine production of TNFα. In other cell types, such as mouse lung fibroblasts and macrophages, Akt exhibited control over necroptosis-associated TNFα production without contributing to cell death. Overall, our results provide new insights into the mechanism of necroptosis and the role of Akt kinase in both cell death and inflammatory regulation. PMID:23469174

  1. Akt Regulates TNFα synthesis downstream of RIP1 kinase activation during necroptosis.

    PubMed

    McNamara, Colleen R; Ahuja, Ruchita; Osafo-Addo, Awo D; Barrows, Douglas; Kettenbach, Arminja; Skidan, Igor; Teng, Xin; Cuny, Gregory D; Gerber, Scott; Degterev, Alexei

    2013-01-01

    Necroptosis is a regulated form of necrotic cell death that has been implicated in the pathogenesis of various diseases including intestinal inflammation and systemic inflammatory response syndrome (SIRS). In this work, we investigated the signaling mechanisms controlled by the necroptosis mediator receptor interacting protein-1 (RIP1) kinase. We show that Akt kinase activity is critical for necroptosis in L929 cells and plays a key role in TNFα production. During necroptosis, Akt is activated in a RIP1 dependent fashion through its phosphorylation on Thr308. In L929 cells, this activation requires independent signaling inputs from both growth factors and RIP1. Akt controls necroptosis through downstream targeting of mammalian Target of Rapamycin complex 1 (mTORC1). Akt activity, mediated in part through mTORC1, links RIP1 to JNK activation and autocrine production of TNFα. In other cell types, such as mouse lung fibroblasts and macrophages, Akt exhibited control over necroptosis-associated TNFα production without contributing to cell death. Overall, our results provide new insights into the mechanism of necroptosis and the role of Akt kinase in both cell death and inflammatory regulation.

  2. The molecular choreography of protein synthesis: translational control, regulation, and pathways.

    PubMed

    Chen, Jin; Choi, Junhong; O'Leary, Seán E; Prabhakar, Arjun; Petrov, Alexey; Grosely, Rosslyn; Puglisi, Elisabetta Viani; Puglisi, Joseph D

    2016-01-01

    Translation of proteins by the ribosome regulates gene expression, with recent results underscoring the importance of translational control. Misregulation of translation underlies many diseases, including cancer and many genetic diseases. Decades of biochemical and structural studies have delineated many of the mechanistic details in prokaryotic translation, and sketched the outlines of eukaryotic translation. However, translation may not proceed linearly through a single mechanistic pathway, but likely involves multiple pathways and branchpoints. The stochastic nature of biological processes would allow different pathways to occur during translation that are biased by the interaction of the ribosome with other translation factors, with many of the steps kinetically controlled. These multiple pathways and branchpoints are potential regulatory nexus, allowing gene expression to be tuned at the translational level. As research focus shifts toward eukaryotic translation, certain themes will be echoed from studies on prokaryotic translation. This review provides a general overview of the dynamic data related to prokaryotic and eukaryotic translation, in particular recent findings with single-molecule methods, complemented by biochemical, kinetic, and structural findings. We will underscore the importance of viewing the process through the viewpoints of regulation, translational control, and heterogeneous pathways.

  3. Live attenuated Salmonella enterica serovar Choleraesuis vaccine vector displaying regulated delayed attenuation and regulated delayed antigen synthesis to confer protection against Streptococcus suis in mice.

    PubMed

    Ji, Zhenying; Shang, Jing; Li, Yuan; Wang, Shifeng; Shi, Huoying

    2015-09-11

    Salmonella enterica serotype Choleraesuis (S. Choleraesuis) and Streptococcus suis (S. suis) are important swine pathogens. Development of a safe and effective attenuated S. Choleraesuis vaccine vector would open a new window to prevent and control pig diseases. To achieve this goal, the mannose and arabinose regulated delayed attenuated systems (RDAS), Δpmi and ΔPcrp::TT araC PBADcrp, were introduced into the wild type S. Choleraesuis strain C78-3. We also introduced ΔrelA::araC PBADlacI TT to achieve regulated delayed antigen synthesis and ΔasdA to constitute a balanced-lethal plasmid system. The safety and immunogenicity of the resulted RDAS S. Choleraesuis strain rSC0011 carrying 6-phosphogluconate dehydrogenase (6-PGD) of S. suis serotype 2 (SS2) were evaluated in vitro and in vivo. Compared with the wild type parent strain C78-3 and vaccine strain C500, a live attenuated S. Choleraesuis vaccine licensed for piglet in China, the results showed that the survival curves of the vaccine strain rSC0011 were similar to those of strains C78-3 and C500 at the early stage of infection, but lower than those of C78-3 and higher than those of C500 at the later stage in both porcine alveolar macrophages and peripheral porcine monocytes. The LD50 of the RDAS strains rSC0011 by oral route in mice was close to that of C500 and 10,000-fold higher than that of C78-3. Similar results were achieved by intraperitoneal (i.p.) route, suggesting that the RDAS strains rSC0011 achieved similar attenuation as C500. However, the RDAS strain rSC0011 was superior to C500 in colonization of Peyer's patches. Adult mice orally immunized with strain rSC0011 carrying a plasmid expression 6-phosphogluconate dehydrogenase (6-PGD) gene from SS2 developed strong immune responses against 6-PGD and Salmonella antigens, and conferred high protection against i.p. challenge with SS2.

  4. Downregulation of the autophagy protein ATG-7 correlates with distinct sphingolipid profile in MCF-7 cells sensitized to photodamage

    NASA Astrophysics Data System (ADS)

    Separovic, Duska; Kelekar, Ameeta; Tarca, Adi L.; Bielawski, Jacek; Kessel, David

    2009-06-01

    The objective of this study was to determine the sphingolipid (SL) profile in autophagy-defective cells and overall cell death after PDT with Pc 4 (PDT). Human breast cancer MCF-7 cells with downregulated autophagy protein ATG-7 and their scrambled controls (Scr) were used. Exposure of ATG-7 knockdown cells to PDT led to defective processing of the autophagy marker LC3, and increased overall cell killing. In both cell types PDT evoked an early (2 h) increase in ceramides and dihydroceramides (DHceramides). When the two cell types were compared regarding time (2 and 24 h) and treatment conditions (with and without PDT), the levels of several ceramides and DHceramides were reduced, whereas the concentrations of C14-ceramide, C16-ceramide and C12-DHceramide were higher in ATG-7 knockdown cells. The data imply that the SL profile might be a marker of autophagy-deficiency in cells sensitized to PDT.

  5. Requirement for the eIF4E binding proteins for the synergistic down-regulation of protein synthesis by hypertonic conditions and mTOR inhibition.

    PubMed

    Clemens, Michael J; Elia, Androulla; Morley, Simon J

    2013-01-01

    The protein kinase mammalian target of rapamycin (mTOR) regulates the phosphorylation and activity of several proteins that have the potential to control translation, including p70S6 kinase and the eIF4E binding proteins 4E-BP1 and 4E-BP2. In spite of this, in exponentially growing cells overall protein synthesis is often resistant to mTOR inhibitors. We report here that sensitivity of wild-type mouse embryonic fibroblasts (MEFs) to mTOR inhibitors can be greatly increased when the cells are subjected to the physiological stress imposed by hypertonic conditions. In contrast, protein synthesis in MEFs with a double knockout of 4E-BP1 and 4E-BP2 remains resistant to mTOR inhibitors under these conditions. Phosphorylation of p70S6 kinase and protein kinase B (Akt) is blocked by the mTOR inhibitor Ku0063794 equally well in both wild-type and 4E-BP knockout cells, under both normal and hypertonic conditions. The response of protein synthesis to hypertonic stress itself does not require the 4E-BPs. These data suggest that under certain stress conditions: (i) translation has a greater requirement for mTOR activity and (ii) there is an absolute requirement for the 4E-BPs for regulation by mTOR. Importantly, dephosphorylation of p70S6 kinase and Akt is not sufficient to affect protein synthesis acutely.

  6. Regulation of capsule synthesis and cell motility in Salmonella enterica by the essential gene igaA.

    PubMed Central

    Cano, David A; Domínguez-Bernal, Gustavo; Tierrez, Alberto; Garcia-Del Portillo, Francisco; Casadesús, Josep

    2002-01-01

    Mutants of Salmonella enterica carrying the igaA1 allele, selected as able to overgrow within fibroblast cells in culture, are mucoid and show reduced motility. Mucoidy is caused by derepression of wca genes (necessary for capsule synthesis); these genes are regulated by the RcsC/YojN/RcsB phosphorelay system and by the RcsA coregulator. The induction of wca expression in an igaA1 mutant is suppressed by mutations in rcsA and rcsC. Reduced motility is caused by lowered expression of the flagellar master operon, flhDC, and is suppressed by mutations in rcsB or rcsC, suggesting that mutations in the igaA gene reduce motility by activating the RcsB/C system. A null igaA allele can be maintained only in an igaA(+)/igaA merodiploid, indicating that igaA is an essential gene. Lethality is suppressed by mutations in rcsB, rcsC, and yojN, but not in rcsA, suggesting that the viability defect of an igaA null mutant is mediated by the RcsB/RcsC system, independently of RcsA (and therefore of the wca genes). Because all the defects associated with igaA mutations are suppressed by mutations that block the RcsB/RcsC system, we propose a functional interaction between the igaA gene product and either the Rcs regulatory network or one of its regulated products. PMID:12524328

  7. Maize endosperm-specific transcription factors O2 and PBF network the regulation of protein and starch synthesis

    PubMed Central

    Zhang, Zhiyong; Zheng, Xixi; Yang, Jun; Messing, Joachim; Wu, Yongrui

    2016-01-01

    The maize endosperm-specific transcription factors opaque2 (O2) and prolamine-box binding factor (PBF) regulate storage protein zein genes. We show that they also control starch synthesis. The starch content in the PbfRNAi and o2 mutants was reduced by ∼5% and 11%, respectively, compared with normal genotypes. In the double-mutant PbfRNAi;o2, starch was decreased by 25%. Transcriptome analysis reveals that >1,000 genes were affected in each of the two mutants and in the double mutant; these genes were mainly enriched in sugar and protein metabolism. Pyruvate orthophosphate dikinase 1 and 2 (PPDKs) and starch synthase III (SSIII) are critical components in the starch biosynthetic enzyme complex. The expression of PPDK1, PPDK2, and SSIII and their protein levels are further reduced in the double mutants as compared with the single mutants. When the promoters of these genes were analyzed, we found a prolamine box and an O2 box that can be additively transactivated by PBF and O2. Starch synthase IIa (SSIIa, encoding another starch synthase for amylopectin) and starch branching enzyme 1 (SBEI, encoding one of the two main starch branching enzymes) are not directly regulated by PBF and O2, but their protein levels are significantly decreased in the o2 mutant and are further decreased in the double mutant, indicating that o2 and PbfRNAi may affect the levels of some other transcription factor(s) or mRNA regulatory factor(s) that in turn would affect the transcript and protein levels of SSIIa and SBEI. These findings show that three important traits—nutritional quality, calories, and yield—are linked through the same transcription factors. PMID:27621432

  8. Differential Sphingolipid and Phospholipid Profiles in Alcohol and Nicotine-Derived Nitrosamine Ketone (NNK) Associated White Matter Degeneration

    PubMed Central

    Yalcin, Emine B.; Nunez, Kavin; Tong, Ming; de la Monte, Suzanne M.

    2015-01-01

    Background Alcohol-mediated neurodegeneration is associated with white matter (WM) atrophy due to targeting of myelin and oligodendrocytes. However, variability in disease severity suggests co-factors contribute to WM degeneration. We examined the potential co-factor role of the tobacco-specific nitrosamine, nicotine-derived nitrosamine ketone (NNK), since smoking causes WM atrophy and most heavy drinkers consume tobacco products. Methods This 8-week study of Long Evans rats had 4 treatment groups: control; NNK-2 mg/kg, 3×/wk in Wks 3–8; ethanol (chronic-26% caloric + binge-2 g/kg, 3×/week in Wks 7–8); and ethanol+NNK. Exposure effects on WM lipid biochemical profiles and in situ distributions were examined using matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) and tandem mass spectrometry. Results NNK mainly caused WM fiber degeneration and fiber loss, ethanol caused demyelination, and dual exposures had additive effects. Ethanol and ethanol+NNK decreased WM (including corpus callosum) and/or gray matter (hypothalamus, cortex, medial temporal) levels of several phosphatidylserine (PS), phosphatidylinositol (PI) and sphingolipid (sulfatide; ST) species, while NNK increased or had minimal effect on these lipids. Ethanol+NNK had broader and larger inhibitory effects on phospholipids and sulfatides than ethanol or NNK alone. Principle component analysis clustered control with NNK, and ethanol with ethanol+NNK groups, highlighting the independent ethanol-rather than NNK-driven responses. Conclusion Chronic ethanol exposures decreased several phospholipid and sphingolipid species in brain, while concomitant NNK exposures exacerbated these effects. These findings support our hypothesis that tobacco smoking is a pathogenic co-factor in alcohol-mediated WM degeneration. PMID:26756797

  9. The Synthetic Amphipathic Peptidomimetic LTX109 Is a Potent Fungicide That Disturbs Plasma Membrane Integrity in a Sphingolipid Dependent Manner

    PubMed Central

    Larsen, Camilla Eggert; Folkesson, Anders; Regenberg, Birgitte

    2013-01-01

    The peptidomimetic LTX109 (arginine-tertbutyl tryptophan-arginine-phenylethan) was previously shown to have antibacterial properties. Here, we investigated the activity of this novel antimicrobial peptidomimetic on the yeast Saccharomyces cerevisiae. We found that LTX109 was an efficient fungicide that killed all viable cells in an exponentially growing population as well as a large proportion of cells in biofilm formed on an abiotic surface. LTX109 had similar killing kinetics to the membrane-permeabilizing fungicide amphotericin B, which led us to investigate the ability of LTX109 to disrupt plasma membrane integrity. S. cerevisiae cells exposed to a high concentration of LTX109 showed rapid release of potassium and amino acids, suggesting that LTX109 acted by destabilizing the plasma membrane. This was supported by the finding that cells were permeable to the fluorescent nucleic acid stain SYTOX Green after a few minutes of LTX109 treatment. We screened a haploid S. cerevisiae gene deletion library for mutants resistant to LTX109 to uncover potential molecular targets. Eight genes conferred LTX109 resistance when deleted and six were involved in the sphingolipid biosynthetic pathway (SUR1, SUR2, SKN1, IPT1, FEN1 and ORM2). The involvement of all of these genes in the biosynthetic pathway for the fungal-specific lipids mannosylinositol phosphorylceramide (MIPC) and mannosyl di-(inositol phosphoryl) ceramide (M(IP)2C) suggested that these lipids were essential for LTX109 sensitivity. Our observations are consistent with a model in which LTX109 kills S. cerevisiae by nonspecific destabilization of the plasma membrane through direct or indirect interaction with the sphingolipids. PMID:23874964

  10. The hypoxic regulator of sterol synthesis Nro1 is a nuclear import adaptor

    PubMed Central

    Yeh, Tzu-Lan; Lee, Chih-Yung S.; Amzel, L. Mario; Espenshade, Peter J.; Bianchet, Mario A.

    2011-01-01

    SUMMARY Fission yeast protein Sre1, the homolog of the mammalian sterol regulatory element binding protein (SREBP), is a hypoxic transcription factor required for sterol homeostasis and low oxygen growth. Nro1 regulates the stability of the N-terminal transcription factor domain of Sre1 (Sre1N) by inhibiting the action of the prolyl 4-hydroxylase-like Ofd1 in an oxygen-dependent manner. The crystal structure of Nro1 determined at 2.2 Å resolution shows an all-α-helical fold that can be divided into two domains: a small N-terminal domain and a larger C-terminal HEAT-repeat domain. Follow-up studies showed that Nro1 defines a new class of nuclear import adaptor that functions both in Ofd1 nuclear localization and in the oxygen-dependent inhibition of Ofd1 to control the hypoxic response. PMID:21481773

  11. The Hypoxic Regulator of Sterol Synthesis Nro1 Is a Nuclear Import Adaptor

    SciTech Connect

    T Yeh; C Lee; L Amzel; P Espenshade; M Bianchet

    2011-12-31

    Fission yeast protein Sre1, the homolog of the mammalian sterol regulatory element-binding protein (SREBP), is a hypoxic transcription factor required for sterol homeostasis and low-oxygen growth. Nro1 regulates the stability of the N-terminal transcription factor domain of Sre1 (Sre1N) by inhibiting the action of the prolyl 4-hydroxylase-like Ofd1 in an oxygen-dependent manner. The crystal structure of Nro1 determined at 2.2 {angstrom} resolution shows an all-{alpha}-helical fold that can be divided into two domains: a small N-terminal domain, and a larger C-terminal HEAT-repeat domain. Follow-up studies showed that Nro1 defines a new class of nuclear import adaptor that functions both in Ofd1 nuclear localization and in the oxygen-dependent inhibition of Ofd1 to control the hypoxic response.

  12. Regulation of the synthesis of barley aleurone. cap alpha. -amylase by gibberellic acid and calcium ions

    SciTech Connect

    Jones, R.L.; Carbonell, J.

    1984-09-01

    The effects of gibberellic acid (GA/sub 3/) and calcium ions on the production of ..cap alpha..-amylase and acid phosphatase by isolated aleurone layers of barley (Hordeum vulgare L. cv Himalaya) were studied. Aleurone layers not previously exposed to GA/sub 3/ or CA/sup 2 +/ show qualitative and quantitative changes in hydrolase production following incubation in either GA/sub 3/ or CA/sup 2 +/ or both. In cubation in H/sub 2/O or CA/sup 2 +/ results in the production of low levels of ..cap alpha..-amylase or acid phosphatase. The addition of GA/sub 3/ to the incubation medium causes 10- to 20-fold increase in the amounts of these enzymes released from the tissue, and addition of CA/sup 2 +/ at 10 millimolar causes a further 8- to 9-fold increase in ..cap alpha..-amylase release and a 75% increase in phosphatase release. Production of ..cap alpha..-amylase isoenzymes is also modified by the levels of GA/sub 3/ and CA/sup 2 +/ in the incubation medium. ..cap alpha..-amylase 2 is produced under all conditions of incubation, while ..cap alpha..-amylase 1 appears only when layers are incubated in GA/sub 3/ or GA/sub 3/ plus CA/sup 2 +/. The synthesis of ..cap alpha..-amylases 3 and 4 requires the presence of both GA/sub 3/ and CA/sup 2 +/ in the incubation medium. Laurell rocket immunoelectrophoresis shows that two distinct groups of ..cap alpha..-amylase antigens are present in incubation media of aleurone layers incubated with both GA/sub 3/ and CA/sup 2 +/, while only one group of antigens is found in media of layers incubated in GA/sub 3/ alone. Strontium ions can be substituted for CA/sup 2 +/ in increasing hydrolase production, although higher concentrations of Sr/sup 2 +/ are requried for maximal response. We conclude that GA/sub 3/ is required for the production of ..cap alpha..-amylase 1 and that both GA/sub 3/ and either CA/sup 2 +/ or Sr/sup 2 +/ are required for the production of isoenzymes 3 and 4 of barley aleurone ..cap alpha..-amylase. 22 references, 8

  13. Regulation of cyclic AMP synthesis and degradation is modified in rat liver at late gestation.

    PubMed Central

    Martinez, C; Ruiz, P; Satrustegui, J; Andres, A; Carrascosa, J M

    1992-01-01

    Cyclic AMP (cAMP) is known to play a key role in regulating insulin action, and it is well documented that in several cases of physiological insulin resistance its concentration is increased. Since late pregnancy in the rat is associated with liver insulin resistance, we have studied possible alterations of some cellular mechanisms regulating the cAMP metabolism. (1) Liver cAMP concentration was shown to be increased by some 30% and 50% at 18 and 22 days of pregnancy respectively, compared with virgins. (2) Basal adenylate cyclase activity was higher only in the 18-days-pregnant rat, and the forskolin-stimulated maximal activity was similar in the three groups of animals. (3) alpha s protein is decreased in term-pregnant rats; however, coupling between Gs and adenylate cyclase is only impaired in the 18-days-pregnant animals, and stimulation by glucagon is impaired in both groups of pregnant animals. (4) Gi-2 protein was shown to be unable to elicit the tonic inhibition of adenylate cyclase in pregnant rats, although it was only decreased at 22 days of gestation. The increased alpha i-2 level detected by immunoblotting at 18 days of gestation did not correlate with its decreased ADP-ribosylation, suggesting that the protein is somehow modified at this stage. (5) Pregnancy is associated with a decrease in membrane phosphodiesterase activity. Our results show that late pregnancy is associated with increases in liver cAMP levels that might be involved in eliciting the characteristic insulin-resistant state, and suggest that mechanisms leading to these increments are changing during this phase of gestation. Images Fig. 2. Fig. 3. PMID:1326941

  14. Recombinant TAT-Gelonin Fusion Toxin: Synthesis and Characterization of Heparin/Protamine-Regulated Cell Transduction

    PubMed Central

    Zhang, Jian; Huang, Yongzhuo; He, Huining; Wang, Mei; Min, Kyoung Ah; Yang, Victor C.

    2014-01-01

    Protein toxins, such as gelonin, are highly desirable anti-cancer drug candidates due to their unparalleled potency and repetitive reaction mechanism in inhibiting protein translation. However, for its potential application in cancer therapy, there remains the cell membrane barrier that allows permeation of only small molecules, which must be overcome. To address this challenge, we conjugated gelonin with a protein transduction domain (PTD), the TAT peptide, via genetic recombination. The chimeric TAT-gelonin fusion protein (TAT-Gel) retained equipotent N-glycosidase activity yet displayed greater cell uptake than unmodified recombinant gelonin (rGel), thereby yielding a significantly augmented cytotoxic activity. Remarkably, TATGel displayed up to 177-fold lower IC50 (avg. 54.3 nM) than rGel (avg. IC50: 3640 nM) in tested cell lines. This enhanced cytotoxicity, however, also raised potential toxicity concerns due to the non-selectivity of PTD in its mediated cell transduction. To solve this problem, we investigated the plausibility of regulating the cell transduction of TAT-Gel via a reversible masking using heparin and protamine. Here, we demonstrated, both in vitro and in vivo, that the cell transduction of TAT-Gel can be completely curbed with heparin and yet this heparin block can be efficiently reversed by the addition of protamine. This reversible tight regulation of the cell transduction of TAT-Gel by heparin and protamine sheds light of possible application of TATGel in achieving a highly effective yet safe drug therapy for the treatment of tumors. PMID:24733757

  15. Proliferating cell nuclear antigen (PCNA)-binding protein C1orf124 is a regulator of translesion synthesis.

    PubMed

    Ghosal, Gargi; Leung, Justin Wai-Chung; Nair, Binoj C; Fong, Ka-Wing; Chen, Junjie

    2012-10-05

    DNA damage-induced proliferating cell nuclear antigen (PCNA) ubiquitination serves as the key event mediating post-replication repair. Post-replication repair involves either translesion synthesis (TLS) or damage avoidance via template switching. In this study, we have identified and characterized C1orf124 as a regulator of TLS. C1orf124 co-localizes and interacts with unmodified and mono-ubiquitinated PCNA at UV light-induced damage sites, which require the PIP box and UBZ domain of C1orf124. C1orf124 also binds to the AAA-ATPase valosin-containing protein via its SHP domain, and cellular resistance to UV radiation mediated by C1orf124 requires its interactions with valosin-containing protein and PCNA. Interestingly, C1orf124 binds to replicative DNA polymerase POLD3 and PDIP1 under normal conditions but preferentially associates with TLS polymerase η (POLH) upon UV damage. Depletion of C1orf124 compromises PCNA monoubiquitination, RAD18 chromatin association, and RAD18 localization to UV damage sites. Thus, C1orf124 acts at multiple steps in TLS, stabilizes RAD18 and ubiquitinated PCNA at damage sites, and facilitates the switch from replicative to TLS polymerase to bypass DNA lesion.

  16. Two-component systems are involved in the regulation of botulinum neurotoxin synthesis in Clostridium botulinum type A strain Hall.

    PubMed

    Connan, Chloé; Brüggemann, Holger; Brueggemann, Holger; Mazuet, Christelle; Raffestin, Stéphanie; Cayet, Nadège; Popoff, Michel R

    2012-01-01

    Clostridium botulinum synthesizes a potent neurotoxin (BoNT) which associates with non-toxic proteins (ANTPs) to form complexes of various sizes. The bont and antp genes are clustered in two operons. In C. botulinum type A, bont/A and antp genes are expressed during the end of the exponential growth phase and the beginning of the stationary phase under the control of an alternative sigma factor encoded by botR/A, which is located between the two operons. In the genome of C. botulinum type A strain Hall, 30 gene pairs predicted to encode two-component systems (TCSs) and 9 orphan regulatory genes have been identified. Therefore, 34 Hall isogenic antisense strains on predicted regulatory genes (29 TCSs and 5 orphan regulatory genes) have been obtained by a mRNA antisense procedure. Two TCS isogenic antisense strains showed more rapid growth kinetics and reduced BoNT/A production than the control strain, as well as increased bacterial lysis and impairment of the bacterial cell wall structure. Three other TCS isogenic antisense strains induced a low level of BoNT/A and ANTP production. Interestingly, reduced expression of bont/A and antp genes was shown to be independent of botR/A. These results indicate that BoNT/A synthesis is under the control of a complex network of regulation including directly at least three TCSs.

  17. Role of Protein Phosphorylation and Tyrosine Phosphatases in the Adrenal Regulation of Steroid Synthesis and Mitochondrial Function.

    PubMed

    Paz, Cristina; Cornejo Maciel, Fabiana; Gorostizaga, Alejandra; Castillo, Ana F; Mori Sequeiros García, M Mercedes; Maloberti, Paula M; Orlando, Ulises D; Mele, Pablo G; Poderoso, Cecilia; Podesta, Ernesto J

    2016-01-01

    In adrenocortical cells, adrenocorticotropin (ACTH) promotes the activation of several protein kinases. The action of these kinases is linked to steroid production, mainly through steroidogenic acute regulatory protein (StAR), whose expression and activity are dependent on protein phosphorylation events at genomic and non-genomic levels. Hormone-dependent mitochondrial dynamics and cell proliferation are functions also associated with protein kinases. On the other hand, protein tyrosine dephosphorylation is an additional component of the ACTH signaling pathway, which involves the "classical" protein tyrosine phosphatases (PTPs), such as Src homology domain (SH) 2-containing PTP (SHP2c), and members of the MAP kinase phosphatase (MKP) family, such as MKP-1. PTPs are rapidly activated by posttranslational mechanisms and participate in hormone-stimulated steroid production. In this process, the SHP2 tyrosine phosphatase plays a crucial role in a mechanism that includes an acyl-CoA synthetase-4 (Acsl4), arachidonic acid (AA) release and StAR induction. In contrast, MKPs in steroidogenic cells have a role in the turn-off of the hormonal signal in ERK-dependent processes such as steroid synthesis and, perhaps, cell proliferation. This review analyzes the participation of these tyrosine phosphates in the ACTH signaling pathway and the action of kinases and phosphatases in the regulation of mitochondrial dynamics and steroid production. In addition, the participation of kinases and phosphatases in the signal cascade triggered by different stimuli in other steroidogenic tissues is also compared to adrenocortical cell/ACTH and discussed.

  18. Down-regulation of UDP-glucose dehydrogenase affects glycosaminoglycans synthesis and motility in HCT-8 colorectal carcinoma cells

    SciTech Connect

    Wang, Tsung-Pao; Pan, Yun-Ru; Fu, Chien-Yu; Chang, Hwan-You

    2010-10-15

    UDP-glucose dehydrogenase (UGDH) catalyzes oxidation of UDP-glucose to yield UDP-glucuronic acid, a precursor of hyaluronic acid (HA) and other glycosaminoglycans (GAGs) in extracellular matrix. Although association of extracellular matrix with cell proliferation and migration has been well documented, the importance of UGDH in these behaviors is not clear. Using UGDH-specific small interference RNA to treat HCT-8 colorectal carcinoma cells, a decrease in both mRNA and protein levels of UGDH, as well as the cellular UDP-glucuronic acid and GAG production was observed. Treatment of HCT-8 cells with either UGDH-specific siRNA or HA synthesis inhibitor 4-methylumbelliferone effectively delayed cell aggregation into multicellular spheroids and impaired cell motility in both three-dimensional collagen gel and transwell migration assays. The reduction in cell aggregation and migration rates could be restored by addition of exogenous HA. These results indicate that UGDH can regulate cell motility through the production of GAG. The enzyme may be a potential target for therapeutic intervention of colorectal cancers.

  19. Two-Component Systems Are Involved in the Regulation of Botulinum Neurotoxin Synthesis in Clostridium botulinum Type A Strain Hall

    PubMed Central

    Connan, Chloé; Brueggemann, Holger; Mazuet, Christelle; Raffestin, Stéphanie; Cayet, Nadège; Popoff, Michel R.

    2012-01-01

    Clostridium botulinum synthesizes a potent neurotoxin (BoNT) which associates with non-toxic proteins (ANTPs) to form complexes of various sizes. The bont and antp genes are clustered in two operons. In C. botulinum type A, bont/A and antp genes are expressed during the end of the exponential growth phase and the beginning of the stationary phase under the control of an alternative sigma factor encoded by botR/A, which is located between the two operons. In the genome of C. botulinum type A strain Hall, 30 gene pairs predicted to encode two-component systems (TCSs) and 9 orphan regulatory genes have been identified. Therefore, 34 Hall isogenic antisense strains on predicted regulatory genes (29 TCSs and 5 orphan regulatory genes) have been obtained by a mRNA antisense procedure. Two TCS isogenic antisense strains showed more rapid growth kinetics and reduced BoNT/A production than the control strain, as well as increased bacterial lysis and impairment of the bacterial cell wall structure. Three other TCS isogenic antisense strains induced a low level of BoNT/A and ANTP production. Interestingly, reduced expression of bont/A and antp genes was shown to be independent of botR/A. These results indicate that BoNT/A synthesis is under the control of a complex network of regulation including directly at least three TCSs. PMID:22848632

  20. Regulation of glycogen synthesis by the laforin-malin complex is modulated by the AMP-activated protein kinase pathway.

    PubMed

    Solaz-Fuster, Maria Carmen; Gimeno-Alcañiz, José Vicente; Ros, Susana; Fernandez-Sanchez, Maria Elena; Garcia-Fojeda, Belen; Criado Garcia, Olga; Vilchez, David; Dominguez, Jorge; Garcia-Rocha, Mar; Sanchez-Piris, Maribel; Aguado, Carmen; Knecht, Erwin; Serratosa, Jose; Guinovart, Joan Josep; Sanz, Pascual; Rodriguez de Córdoba, Santiago

    2008-03-01

    Lafora progressive myoclonus epilepsy (LD) is a fatal autosomal recessive neurodegenerative disorder characterized by the presence of glycogen-like intracellular inclusions called Lafora bodies. LD is caused by mutations in two genes, EPM2A and EPM2B, encoding respectively laforin, a dual-specificity protein phosphatase, and malin, an E3 ubiquitin ligase. Previously, we and others have suggested that the interactions between laforin and PTG (a regulatory subunit of type 1 protein phosphatase) and between laforin and malin are critical in the pathogenesis of LD. Here, we show that the laforin-malin complex downregulates PTG-induced glycogen synthesis in FTO2B hepatoma cells through a mechanism involving ubiquitination and degradation of PTG. Furthermore, we demonstrate that the interaction between laforin and malin is a regulated process that is modulated by the AMP-activated protein kinase (AMPK). These findings provide further insights into the critical role of the laforin-malin complex in the control of glycogen metabolism and unravel a novel link between the energy sensor AMPK and glycogen metabolism. These data advance our understanding of the functional role of laforin and malin, which hopefully will facilitate the development of appropriate LD therapies.

  1. Role of intramitochondrial arachidonic acid and acyl-CoA synthetase 4 in angiotensin II-regulated aldosterone synthesis in NCI-H295R adrenocortical cell line.

    PubMed

    Mele, Pablo G; Duarte, Alejandra; Paz, Cristina; Capponi, Alessandro; Podestá, Ernesto J

    2012-07-01

    Although the role of arachidonic acid (AA) in angiotensin II (ANG II)- and potassium-stimulated steroid production in zona glomerulosa cells is well documented, the mechanism responsible for AA release is not fully described. In this study we evaluated the mechanism involved in the release of intramitochondrial AA and its role in the regulation of aldosterone synthesis by ANG II in glomerulosa cells. We show that ANG II and potassium induce the expression of acyl-coenzyme A (CoA) thioesterase 2 and acyl-CoA synthetase 4, two enzymes involved in intramitochondrial AA generation/export system well characterized in other steroidogenic systems. We demonstrate that mitochondrial ATP is required for AA generation/export system, steroid production, and steroidogenic acute regulatory protein induction. We also demonstrate the role of protein tyrosine phosphatases regulating acyl-CoA synthetase 4 and steroidogenic acute regulatory protein induction, and hence ANG II-stimulated aldosterone synthesis.

  2. Developmental Regulation of Lectin and Alliinase Synthesis in Garlic Bulbs and Leaves.

    PubMed Central

    Smeets, K.; Van Damme, EJM.; Peumans, W. J.

    1997-01-01

    Using a combination of northern blot analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a detailed study was made of the temporal and spatial regulation of garlic (Allium sativum L.) lectins and alliinase throughout the life cycle of the plant. The two bulb-specific lectins (ASAI and ASAII), which are the most predominant bulb proteins, accumulate exclusively in the developing garlic cloves and progressively disappear when the old clove is consumed by the plant. On the basis of these observations, ASAI and ASAII can be regarded as typical vegetative storage proteins. The leaf-specific lectin (ASAL), on the contrary, is specifically synthesized in young leaves and remains present until withering. Because ASAL is only a minor protein, it probably fulfills a specific function in the plant. Unlike the lectins, alliinase is present in large quantities in bulbs as well as in leaves. Moreover, intact alliinase mRNAs are present in both tissues as long as they contain living cells. The latter observation is in good agreement with the possible involvement of alliinase in the plant's defense against pathogens and/or predators. PMID:12223641

  3. Thiamine synthesis regulates the fermentation mechanisms in the fungus Aspergillus nidulans.

    PubMed

    Shimizu, Motoyuki; Masuo, Shunsuke; Itoh, Eriko; Zhou, Shengmin; Kato, Masashi; Takaya, Naoki

    2016-09-01

    Thiamine pyrophosphate (TPP) is a critical cofactor and its biosynthesis is under the control of TPP availability. Here we disrupted a predicted thiA gene of the fungus Aspergillus nidulans and demonstrated that it is essential for synthesizing cellular thiamine. The thiamine riboswitch is a post-transcriptional mechanism for TPP to repress gene expression and it is located on A. nidulans thiA pre-messenger RNA. The thiA riboswitch was not fully derepressed under thiamine-limited conditions, and fully derepressed under environmental stressors. Upon exposure to hypoxic stress, the fungus accumulated more ThiA and NmtA proteins, and more thiamine than under aerobic conditions. The thiA gene was required for the fungus to upregulate hypoxic branched-chain amino acids and ethanol fermentation that involve enzymes containing TPP. These findings indicate that hypoxia modulates thiA expression through the thiamine riboswitch, and alters cellular fermentation mechanisms by regulating the activity of the TPP enzymes.

  4. The herpes simplex virus 1 U{sub S}3 regulates phospholipid synthesis

    SciTech Connect

    Wild, Peter; Oliveira, Anna Paula de; Sonda, Sabrina; Schraner, Elisabeth M.; Ackermann, Mathias; Tobler, Kurt

    2012-10-25

    Herpes simplex virus type 1 capsids bud at nuclear and Golgi membranes for envelopment by phospholipid bilayers. In the absence of U{sub S}3, nuclear membranes form multiple folds harboring virions that suggests disturbance in membrane turnover. Therefore, we investigated phospholipid metabolism in cells infected with the U{sub S}3 deletion mutant R7041({Delta}U{sub S}3), and quantified membranes involved in viral envelopment. We report that (i) [{sup 3}H]-choline incorporation into nuclear membranes and cytoplasmic membranes was enhanced peaking at 12 or 20 h post inoculation with wild type HSV-1 and R7041({Delta}U{sub S}3), respectively, (ii) the surface area of nuclear membranes increased until 24 h of R7041({Delta}U{sub S}3) infection forming folds that equaled {approx}45% of the nuclear surface, (iii) the surface area of viral envelopes between nuclear membranes equaled {approx}2400 R7041({Delta}U{sub S}3) virions per cell, and (iv) during R7041({Delta}U{sub S}3) infection, the Golgi complex expanded dramatically. The data indicate that U{sub S}3 plays a significant role in regulation of membrane biosynthesis.

  5. The synthesis and application involving regulation of the insoluble drug release from mesoporous silica nanotubes

    NASA Astrophysics Data System (ADS)

    Li, Jia; Wang, Yan; Zheng, Xin; Zhang, Ying; Sun, Changshan; Gao, Yikun; Jiang, Tongying; Wang, Siling

    2015-03-01

    Mesoporous silica nanotubes (SNT) were synthesized using hard template carbon nanotubes (CNT) with the aid of cetyltrimethyl ammonium bromide (CTAB) in a method, which was simple and inexpensive. Scanning electron microscopy, transmission electron microscopy and specific surface area analysis were employed to characterize the morphology and structure of SNT, and the formation mechanism of SNT was also examined by Fourier transform infrared spectroscopy. There are few published reports of the mesoporous SNT with large specific surface area applied in the drug delivery systems to improve the amount of drug loading. In addition, the structure of SNT allows investigators to control the drug particle size in the pore channels and significantly increase the drug dissolution rate. The insoluble drug, cilostazol, was chosen as a model drug to be loaded into SNT and we developed a simple and efficient method for regulating the drug release by using a gelatin coating with different thicknesses around the SNT. The release rate was adjusted by the amount of gelatin surrounding the SNT, with an increased barrier leading to a reduction in the release rate. A model developed on the basis of the Weibull modulus was established to fit the release results.

  6. TOR complex 2-Ypk1 signaling regulates actin polarization via reactive oxygen species.

    PubMed

    Niles, Brad J; Powers, Ted

    2014-12-01

    The evolutionarily conserved mTOR complex 2 (mTORC2) signaling pathway is an important regulator of actin cytoskeletal architecture and, as such, is a candidate target for preventing cancer cell motility and invasion. Remarkably, the precise mechanism(s) by which mTORC2 regulates the actin cytoskeleton have remained elusive. Here we show that in budding yeast, TORC2 and its downstream kinase Ypk1 regulate actin polarization by controlling reactive oxygen species (ROS) accumulation. Specifically, we find that TORC2-Ypk1 regulates actin polarization both by vacuole-related ROS, controlled by the phospholipid flippase kinase Fpk1 and sphingolipids, and by mitochondria-mediated ROS, controlled by the PKA subunit Tpk3. In addition, we find that the protein kinase C (Pkc1)/MAPK cascade, a well-established regulator of actin, acts downstream of Ypk1 to regulate ROS, in part by promoting degradation of the oxidative stress responsive repressor, cyclin C. Furthermore, we show that Ypk1 regulates Pkc1 activity through proper localization of Rom2 at the plasma membrane, which is also dependent on Fpk1 and sphingolipids. Together these findings demonstrate important links between TORC2/Ypk1 signaling, Fpk1, sphingolipids, Pkc1, and ROS as regulators of actin and suggest that ROS may play an important role in mTORC2-dependent dysregulation of the actin cytoskeleton in cancer cells.

  7. Evaluation of the change in sphingolipids in the human multiple myeloma cell line U266 and gastric cancer cell line MGC-803 treated with arsenic trioxide.

    PubMed

    Zou, Jianhua; Ma, Xiaoqiong; Zhang, Guangji; Shen, Li; Zhou, Liting; Yu, Yu; Zhu, Fanfan; Chen, Zhe

    2015-11-01

    Arsenic trioxide (As2O3) has been found to display anticancer activity against many types of tumors and has been developed into an anticancer drug in clinical treatments. Sphingolipids are membrane lipids that participate in many signal transduction pathways. In this paper, the changes in sphingolipids of the human multiple myeloma cell line U266 and the gastric cancer cell line MGC-803 treated with arsenic trioxide were investigated using an HPLC-ESI-MS/MS method. Analytes were separated by an XBridge BEH C8 column used for Cer, HexCer, LacCer and SM chromatographic separation, and a Capcell PAK MG II C18 column was used for Sph, dhSph, S1P and dhS1P chromatographic separation and gradient elution with acetonitrile-water containing 0.1% formic acid as a mobile phase. A tandem mass spectrometer QTrap in SRM mode was employed in combination with RPLC as a detector for quantitative analysis. The ceramide/sphingolipid internal standard (IS) mixture was used to quantify the levels of sphingolipids. The distributions of sphingolipids were found to be different in the human multiple myeloma cell line U266 and the gastric cancer cell line MGC-803. Ceramide (Cer), hexosylceramide (HexCer) and dihexosylceramide (Hex2Cer) levels in U266 cell line are higher than those in MGC-803 cell line. Additionally, sphingomyelin (SM), sphingosine-1-phosphate (S1P) and sphinganine-1-phosphate (dhS1P) levels in the MGC-803 cell line are higher than those in the U266 cell line. When treated with arsenic trioxide (1-5μM iAs(III)(As(III) ions)), the levels of Hex2Cer in the human multiple myeloma cell line U266 decreased, and the levels of S1P and dhS1P in the human gastric cancer cell line MGC-803 decreased. The decrease of Hex2Cer, S1P and dhS1P in the human multiple myeloma cell line U266 and gastric cancer cell line MGC-803 were observed when the concentration of iAs(III) is 1.0μM. Therefore, arsenic trioxide exhibits anti-cancer activity by altering the sphingolipid pathway in the

  8. HupO, a Novel Regulator Involved in Thiosulfate-Responsive Control of HupSL [NiFe]-Hydrogenase Synthesis in Thiocapsa roseopersicina

    PubMed Central

    Nagy, Ildikó K.; Kovács, Kornél L.

    2016-01-01

    [NiFe]-hydrogenases are regulated by various factors to fulfill their physiological functions in bacterial cells. The photosynthetic purple sulfur bacterium Thiocapsa roseopersicina harbors four functional [NiFe]-hydrogenases: HynSL, HupSL, Hox1, and Hox2. Most of these hydrogenases are functionally linked to sulfur metabolism, and thiosulfate has a central role in this organism. The membrane-associated Hup hydrogenases have been shown to play a role in energy conservation through hydrogen recycling. The expression of Hup-type hydrogenases is regulated by H2 in Rhodobacter capsulatus and Cupriavidus necator; however, it has been shown that the corresponding hydrogen-sensing system is nonfunctional in T. roseopersicina and that thiosulfate is a regulating factor of hup expression. Here, we describe the discovery and analysis of mutants of a putative regulator (HupO) of the Hup hydrogenase in T. roseopersicina. HupO appears to mediate the transcriptional repression of Hup enzyme synthesis under low-thiosulfate conditions. We also demonstrate that the presence of the Hox1 hydrogenase strongly influences Hup enzyme synthesis in that hup expression was decreased significantly in the hox1 mutant. This reduction in Hup synthesis could be reversed by mutation of hupO, which resulted in strongly elevated hup expression, as well as Hup protein levels, and concomitant in vivo hydrogen uptake activity in the hox1 mutant. However, this regulatory control was observed only at low thiosulfate concentrations. Additionally, weak hydrogen-dependent hup expression was shown in the hupO mutant strain lacking the Hox1 hydrogenase. HupO-mediated Hup regulation therefore appears to link thiosulfate metabolism and the hydrogenase network in T. roseopersicina. PMID:26801573

  9. Glucagon and cAMP inhibit cholesterol 7alpha-hydroxylase (CYP7A1) gene expression in human hepatocytes: discordant regulation of bile acid synthesis and gluconeogenesis.

    PubMed

    Song, Kwang-Hoon; Chiang, John Y L

    2006-01-01

    The gene encoding cholesterol 7alpha-hydroxylase (CYP7A1) is tightly regulated to control bile acid synthesis and maintain lipid homeostasis. Recent studies in mice suggest that bile acid synthesis is regulated by the fasted-to-fed cycle, and fasting induces CYP7A1 gene expression in parallel to the induction of peroxisome proliferators-activated receptor gamma co-activator 1alpha (PGC-1alpha) and phosphoenolpyruvate carboxykinase (PEPCK). How glucagon regulates CYP7A1 gene expression in the human liver is not clear. Here we show that glucagon and cyclic adenosine monophosphate (cAMP) strongly repressed CYP7A1 mRNA expression in human primary hepatocytes. Reporter assays confirmed that cAMP and protein kinase A (PKA) inhibited human CYP7A1 gene transcription, in contrast to their stimulation of the PEPCK gen